Host-dependent zonulin secretion causes the impairment of the small intestine barrier function after bacterial exposure

GASTROENTEROLOGY 2002;123:1607–1615

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,‡ and ALESSIO FASANO* *Department of Pediatrics and Center for Vaccine Development, University of Maryland School of Medicine, Baltimore, Maryland; ‡Istituto Clinica Pediatrica, Universita’ di Ancona, Ancona, Italy; and §Istituto Burlo Garofalo, Trieste, Italy

Background & Aims: Enteric infections have been implicated in the pathogenesis of both food intolerance and autoimmune diseases secondary to the impairment of the intestinal barrier. On the basis of our recent discovery of zonulin, a modulator of small-intestinal tight junctions, we asked whether microorganisms might induce zonulin secretion and increased small-intestinal permeability. Methods: Both ex vivo mammalian small intestines and intestinal cell monolayers were exposed to either pathogenic or nonpathogenic enterobacteria. Zonulin production and changes in paracellular permeability were monitored in Ussing chambers and microsnapwells. Zonula occludens 1 protein redistribution after bacteria colonization was evaluated on cell monolayers. Results: Small intestines exposed to enteric bacteria secreted zonulin. This secretion was independent of either the species of the small intestines or the virulence of the microorganisms tested, occurred only on the luminal aspect of the bacteria-exposed smallintestinal mucosa, and was followed by a decrease in small-intestinal tissue resistance (transepithelial electrical resistance). The transepithelial electrical resistance decrement was secondary to the zonulin-induced tight junction disassembly, as also shown by the disengagement of the protein zonula occludens 1 protein from the tight junctional complex. Conclusions: This zonulindriven opening of the paracellular pathway may represent a defensive mechanism, which flushes out microorganisms and contributes to the host response against bacterial colonization of the small intestine.

he intestinal epithelium is the largest epithelial mucosal layer and provides an interface between the external environment and the mammalian host.1 Healthy, mature small-intestinal mucosa, with its intact tight junctions, serves as the main barrier to the passage of macromolecules. Further, it functions as the major organ of defense against foreign antigens, toxins, and macromolecules entering the host via the oral/enteric route.2 Under physiological conditions, small but immunologically active antigens transverse the intestinal bar-

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rier,3 resulting in antigen-specific immune responses.4 This passage occurs through the paracellular pathway and involves a subtle but sophisticated regulation of intercellular tight junctions that leads to antigen tolerance.5,6 When the integrity of the tight junctions system is compromised, such as in preterm babies or after exposure to radiation, chemotherapy, and/or toxins,7 an immune response to environmental antigens may develop. Whereas tight-junction structure and function have been thoroughly investigated, their physiological regulation has been only partially elucidated.8 We have recently described a novel protein, zonulin,9 a eukaryotic analogue of the Vibrio cholerae– derived zonula occludens toxin (Zot),10 which modulates small-intestinal tightjunction permeability through a protein kinase C ␣–mediated actin polymerization.11 In the absence of enteric infections, the mammalian proximal small intestine is virtually sterile. The colonization of the small intestine by enteric microorganisms (even without apparent mucosal damage or elaboration of specific toxins) typically leads to a more permeable intestine that permits the passage of macromolecules and antigens that may cause immune-mediated pathologic conditions.12 To date, there is no clear explanation for the disturbed physiological regulation of the intestinal permeability secondary to proximal bacterial contamination. In this study, we tested both nonpathogenic and pathogenic bacteria for their ability to induce zonulin secretion by the mammalian gut (small intestine and colon). In an attempt to address species specificity, we used nonpathogenic bacteria as potential inducers of zonulin release in ex vivo experiments by use of intestinal segments from multiple mammalian species, including nonhuman priAbbreviations used in this paper: DMEM, Dulbecco’s modified Eagle medium; ELISA, enzyme-linked immunosorbent assay; TEER, transepithelial electrical resistance; TNF, tumor necrosis factor; ZO1, zonula occludens 1 protein; Zot, zonula occludens toxin. © 2002 by the American Gastroenterological Association 0016-5085/02/$35.00 doi:10.1053/gast.2002.36578

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mates. We developed a new micro-snapwell system to monitor the interaction between bacteria and enterocytes, as well as the release of zonulin from the apical and/or the basolateral side of the small intestine. To establish whether enterocytes are the source of zonulin release, we conducted additional experiments with human intestinal cell lines.

Materials and Methods Bacterial Strains To investigate the interactions between microorganisms and the intestinal mucosa, we chose 4 bacterial strains: (1) Escherichia coli strain 6-1, a prototype of the normal intestinal bacterial flora isolated from the stool of a healthy premature infant and probe-negative for established E. coli virulence factors13; (2) DH5␣, a laboratory E. coli K-12 strain produced by genetic engineering as a prototype of innocuous bacteria14; (3) a virulent Salmonella typhimurium strain SO1344, as a prototype of an enteroinvasive microorganism15; and (4) a virulent E. coli strain 21-1 isolated from an infant affected by necrotizing enterocolitis.16 All tested bacteria neither harbored the zot gene nor secreted Zot in their culture supernatants, as established by enzyme-linked immunosorbent assay (ELISA). For each experiment, fresh bacterial cultures were grown in L-agar overnight at 37°C from frozen stocks.

Organ Culture Technique We used the system described by Browning and Trier17 and Jos et al.,18 with minor modifications. Intestinal explants (proximal jejunum and terminal ileum) were isolated from either adult Wistar rats (weighing 250 –300 g) or adult New Zealand White rabbits (weighing 2–3.5 kg). The segments were opened along the mesenteric border, washed in phosphate-buffered saline (PBS), cut in 5 ⫻ 7 mm pieces (surface area of 37.2 ⫾ 3 mm2), immediately placed on a sterile foam sponge support previously soaked with the culture medium, and transferred to Petri dishes containing 65% Dulbecco’s modified Eagle medium (DMEM; Gibco, Rockville, MD), 20% NCTC 135 (Sigma, St. Louis, MO), 15% fetal calf serum, and insulin (32.5 mg/L). In each dish, the level of the culture medium was adjusted to cover the villous surface. Replicates of jejunum and ileum were incubated with 108 colony-forming units per milliliter, placed in an airtight container under 95% oxygen and 5% CO2 , and incubated on a rocking table at 37°C for 24 hours. Culture medium aliquots were collected at increasing time intervals up to 24 hours for zonulin measurement (see below).

Zonulin ELISA Zonulin concentration was evaluated with a sandwich ELISA. Plastic microtiter plates (Costar, Cambridge, MA) were coated with polyclonal rabbit zonulin-specific anti-Zot antibodies9,19 (dilution 1:100) overnight at 4°C and then blocked by incubation with 0.1% PBS–Tween 20 for 15

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minutes at room temperature. A standard curve was obtained with several dilutions of zonulin (0.78 –50 ng/mL) in 0.05% PBS–Tween 20. Equal aliquots of each standard and test sample were pipetted into the wells and incubated for 1 hour at room temperature. Unbound zonulin was washed out, and the wells were then incubated with agitation with biotinylated anti-Zot antibodies for 1 hour at room temperature. A color reaction was developed by adding 100 ␮L of Extra-Avidin (Sigma) diluted 1:20,000 in 0.1 mol/L of Tris-HCl, 1 mmol/L of MgCl2 , and bovine serum albumin 1% (pH 7.3) for 15 minutes, followed by incubation with 100 ␮L of a solution containing 1 mg/mL of p-nitrophenyl-phosphate substrate (Sigma). Absorbance was read after 30 minutes in a spectrophotometer at 405 nm. To define the intra- and interassay precision of the ELISA–sandwich method, the coefficient of variation was calculated by using 3 replicates from 2 samples with different concentrations of zonulin on 3 consecutive days. The interassay test of the ELISA–sandwich method produced coefficient of variation values of 9.8%. The coefficient of variation of the intra-assay test was 4.2% at day 1, 3.3% at day 2, and 2.9% at day 3. Zonulin concentration was expressed as nanograms per milligram of total protein detected in the culture supernatants.

Western Immunoblotting Analysis To confirm the ELISA data, the organ culture supernatants were analyzed for the presence of zonulin by immunoblotting with zonulin-specific anti-Zot antibodies. On the basis of the ELISA results, samples containing increasing zonulin concentrations were subjected to sodium dodecyl sulfate polyacrylamide gel electrophoresis under reducing conditions and immunoblotted as we have previously described.9,19,20

The Micro-Snapwell System Costar snapwells (Costar Corning Inc., Acton, MA) were modified to attain a reduced surface area (7 vs. 113 mm2) of exposed mucosa to perform experiments on smaller specimens, such as human biopsy samples obtained during upperintestinal endoscopies. A 3-mm-diameter central hole was cut in circular Plexiglas (Rohm & Haas Co., Philadelphia, PA) pieces with a 12-mm diameter. The Plexiglas inserts were washed in 100% ethanol, air-dried, and sterilized overnight under ultraviolet light. Intestinal segments were removed, opened along the mesenteric border, and rinsed free of the intestinal content by using PBS; unstripped pieces of 3.5 mm in diameter were placed on presterilized Whatman paper with the mucosal side oriented upward under a dissecting microscope. Tissues so prepared were then sandwiched between 2 Plexiglas inserts, introduced into Costar snapwells, and placed in the incubator (37°C, 5% CO2) for 30 minutes to stabilize the pH.

Transepithelial Electrical Resistance After 30 minutes of incubation at 37°C and pH stabilization, the transepithelial electrical resistance (TEER) was measured in the micro-snapwell system at increasing time

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intervals after bacterial exposure by using a planar electrode (Endohm SNAP electrode attached to an Evom-G WPI analyzer; World Precision Instruments, Sarasota, FL) and expressed in ohms per square centimeter. The difference between TEER at time 0 and TEER at indicated time points was expressed as ⌬-TEER.

Intestinal Permeability Study in the Micro-Snapwell System Segments of small intestine (jejunum and ileum) of either rabbit or rhesus monkey (7-mm2 exposed surface area) were mounted onto the micro-snapwell system, and their luminal side was incubated for 3 hours with 108 bacteria per milliliter. After TEER was measured at time 0 and at 3 hours, samples were collected from both the mucosal and the serosal sides for zonulin analysis. Uninfected tissues were used as controls. 3H-Inulin was concurrently used as a marker for tight-junction competency. Ten microliters of 3H-inulin was added on the luminal side at time 0, and specimens were collected from the serosal side at increasing time intervals for measurement of the probe passage. Radioactivity was measured in a scintillation ␤ counter. The addition of FZI/0 (1 ␮g/mL) (Biopolymer Laboratories, University of Maryland, Baltimore, MD), a synthetic peptide that mimics the zonulin binding domain and, therefore, blocks the zonulin permeating effect,19,21 was tested to establish whether the effect of bacteria exposure on TEER and inulin passage was zonulin dependent. A scrambled peptide (FZI/1) at the same concentration of FZI/0 (1 ␮g/mL) was used as a control.19 In selected experiments, tumor necrosis factor (TNF)–␣ (Research Diagnostic, Inc., Flanders, NJ) was added to the serosal side of small intestines mounted in the micro-snapwell system, and TEER changes and zonulin release were measured both under basal conditions and in the presence of FZI/0.

Zonulin Release by Cultured Epithelial Cells Both human (Caco2) and rat-derived (IEC6) intestinal cells (passages 25– 40) were cultured from frozen stocks in DMEM supplemented with 1% nonessential amino acids, 1% L-glutamine, 1% sodium pyruvate, 10% fetal calf serum, 100 U of penicillin, and 100 ␮g of streptomycin per milliliter in a 5% CO2 atmosphere at 37°C in 6-well tissue culture plates. After confluence, the cells were allowed to grow for an additional 10 days for cell differentiation.22,23 Medium was changed for both cell lines every third day. Monolayers were then washed 2 times with PBS and incubated with DMEM without antibiotics. Cells were finally exposed to 108 bacteria per milliliter and incubated at 37°C for an additional 5 hours, and culture supernatants were collected for zonulin measurement.

Ussing Chamber Assay Zonulin obtained from organ culture supernatants was tested on rhesus monkey small intestine mounted in Ussing chambers.11 TEER was measured every 10 minutes after the luminal addition of zonulin-containing organ culture superna-

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tants (at a final concentration of 1 ng/mL), either alone or in combination with FZI/0 (1 ␮g/mL).

Direct Immunofluorescence on Epithelial Cell Monolayers and Zonula Occludens (ZO1) Localization/Migration Caco2 and EIC6 cells were grown on 8-chamber slides by using the growth conditions outlined previously and were incubated with E. coli 6-1 in the presence or absence of FZI/0 (1 ␮g/mL) at increasing time intervals. Uninfected monolayers were used as controls. Cells were then washed gently with PBS and permeated with methanol at ⫺20°C for 2 minutes. The monolayers were then washed 3 times with PBS and incubated with primary antibodies (fluorescein isothiocyanate– conjugated anti-ZO1 monoclonal antibody; Zymed Laboratories Inc., San Francisco, CA). After either 30 minutes or 4 hours of incubation, the slides were washed twice with PBS, air-dried, and blindly analyzed by 2 independent observers with a fluorescence microscope (Optiplot; Nikon Inc., Melville, NY).

Statistical Analysis Data are expressed as mean ⫾ SEM. Differences between means were analyzed by 1-way analysis of variance and the Bonferroni test for post hoc multiple comparison. All procedures were performed by the SPSS 10.0 statistical package (SPSS Inc., Chicago, IL).

Results Effect of Enteric Bacteria on Rabbit Intestine Mounted in the Micro-Snapwell System Luminal exposure of rabbit jejunum (data not shown) and ileum (Figure 1A) to both S. typhimurium and pathogenic E. coli 21-1 induced a significant TEER decrease after 3 hours of incubation as compared with the noninfected negative control tissues (P ⫽ 0.013). A smaller but still significant (P ⫽ 0.041) TEER decrease was also detected when rabbit small intestines were incubated with the nonpathogenic E. coli strain 6-1. These changes in TEER correlated with increases in zonulin secretion into the luminal (but not the serosal) side of the mucosa (Figure 1B), where the zonulin receptor has been localized.10,21 Conversely, when E. coli 6-1 was tested on rabbit colonic mucosa, no differences in either TEER (⌬-TEER, 56.0 ⫾ 10.54 ⍀/cm2) or zonulin release (2.29 ng/mg protein) were observed when compared with media-negative controls (⌬-TEER, ⫺38 ⫾ 15.7 ⍀/cm2; zonulin release, 2.46 ng/mg protein). To selectively investigate the primary mammalian small-intestinal response to bacterial colonization, all subsequent experiments were performed with nonpathogenic bacteria (human isolate E. coli strain 6-1 and/or E. coli DH5␣).

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Figure 2. Kinetics of zonulin release from rabbit small-intestine organ cultures either uninfected (Œ) or inoculated with the nonpathogenic bacteria E. coli 6-1 (E) or DH5␣ (●). 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.

Effect of Nonpathogenic E. coli 6-1 on Different Mammalian Ileal Tissues Mounted in the Micro-Snapwell System

Figure 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 ⍀/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.

The experiments described previously were repeated by using small-intestinal tissues obtained from other mammals, including rats and monkeys. As shown in Table 1, nonpathogenic E. coli 6-1 induced changes in both TEER and zonulin secretion, irrespective of the species of intestines challenged. No significant changes of these parameters were observed in uninfected controls (Figure 1; Table 1). Effect of Prolonged Intestinal Exposure to Bacteria on Zonulin Secretion Organ cultures of rabbit small intestines showed a time-dependent secretion of zonulin induced by nonpathogenic E. coli bacteria (Figure 2). Both E. coli strain 6-1 and E. coli DH5␣ induced a significant increase in zonulin release as compared with uninfected controls starting 2 hours after bacteria exposure and continuing up to 48 hours after infection. These changes were paralleled by a TEER decrease in infected tissues at 24 hours (⌬-TEER, ⫺240.0 ⫾ 11.4 ⍀/cm2) and 48 hours (⌬-

Table 1. Effect of Nonpathogenic E. coli 6-1 on Mammal Small-Intestinal Mucosa Mounted in Micro-Snapwells Rat

Rabbit

Monkey

Variable

Zonulin (ng/mg protein)

⌬ TEER (⍀/cm2)

Zonulin (ng/mg protein)

⌬ TEER (⍀/cm2)

Zonulin (ng/mg protein)

⌬ TEER (⍀/cm2)

E. coli 6-1a Uninfected

0.88 ⫾ 0.06 0.004 ⫾ 0.002

⫺34.5 ⫾ 8.3 ⫺8.1 ⫾ 6.6

1.60 ⫾ 0.26 0.015 ⫾ 0.002

⫺33.6 ⫾ 9.9 ⫺6.2 ⫾ 1.07

13.0 ⫾ 2.0 2.97 ⫾ 2.01

⫺78.0 ⫾ 4.5 ⫺31.6 ⫾ 4.4

aP

between ⬍0.05 and ⬍0.01.

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Effect of Nonpathogenic E. coli Strain 6-1 on Rhesus Monkey Small-Intestinal Barrier Functions

Figure 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).

TEER, ⫺310.0 ⫾ 13.5 ⍀/cm2) as compared with uninfected controls (⌬-TEER, ⫺54.0 ⫾ 10.6 ⍀/cm2). Immunoblotting of Organ Culture Supernatants Organ culture supernatants containing increasing zonulin concentrations (as established by ELISA) were subjected to Western blot analysis. As shown in Figure 3, a 47-kilodalton band consistent with the predicted zonulin molecular size9,19 was detected when supernatants containing zonulin at concentrations higher than 13.4 ng/mg of total protein were tested. The density of the immunoreactive band correlated with zonulin concentration in the supernatant tested. Two additional immunoreactive bands of approximately 46 and 20 kilodaltons were also detected.

With the micro-snapwell system, E. coli strain 6-1 induced a significant decrease in TEER after 4 hours when added to either the monkey jejunum or ileum as compared with uninfected small intestine (P ⬍ 0.01) (Table 2). The involvement of the paracellular pathway was confirmed by increased flux of the paracellular marker 3H-inulin from the mucosal to the serosal side in the bacteria-exposed tissues (P ⬍ 0.001) (Figure 4A). The impairment of the barrier function was paralleled by a significant luminal (but not basolateral) secretion of zonulin from the tissues incubated with E. coli 6-1 (P ⬍ 0.001) (Figure 4B). No detectable zonulin was found in supernatants from uninfected small-intestinal segments (Figure 4B). Similar results were obtained when intestinal tissues were exposed to E. coli DH5␣ (data not shown). Pretreatment with FZI/0 (1 ␮g/mL), a zonulin receptor binding inhibitor that prevents the zonulin permeating effect,19 completely blocked the E. coli 6-1– induced TEER changes in both jejunum and ileum (Table 2) and the inulin paracellular passage (Figure 4A) without affecting zonulin luminal secretion (Figure 4B). No inhibitory effects on either TEER changes (Table 2) or inulin passage (Figure 4A) were detected when a scrambled peptide (FZI/1) (1 ␮g/mL) was used. The specificity of the FZI/0 zonulin inhibitory effect was further evaluated by using the cytokine TNF-␣ (2.5 ng/mL) as an alternative stimulus for tight-junction disassembly.22 As expected, TNF-␣ added to the serosal side of the micro-snapwell device caused a significant decrease in TEER, with a peak at 240 minutes after exposure (⌬-TEER, ⫺181.0 ⫾ 12.6 ⍀/cm2 vs. control ⌬-TEER, ⫺42.0 ⫾ 5.2 ⍀/cm2; P ⬍ 0.01). The effect of TNF-␣ on TEER was not mediated by zonulin release on the mucosal side of the tissue (2.43 ng/mg of protein in TNF-treated intestines vs. 1.73 ng/mg of protein in controls; P ⫽ not significant), nor was it blocked by preincubation with FZI/0 1 ␮g/mL (⌬-TEER, ⫺160 ⫾ 20.7 ⍀/cm2).

Table 2. Effect of E. coli 6-1 Infection on Monkey Small Intestines Mounted in the Micro-Snapwell System ⌬-TEER (⍀/cm2) Variable

Uninfected controls

E. coli 6-1

E. coli 6-1 ⫹ FZI/0

E. coli 6-1 ⫹ FZI/1

Monkey jejunum Monkey ileum

⫺28.6 ⫾ 1.2 ⫺31.6 ⫾ 4.4

⫺73.0 ⫾ ⫺78 ⫾ 4.25a

⫺35.0 ⫾ 6.3 ⫺8.6 ⫾ 14.3

⫺85.0 ⫾ 6.9a ⫺81.0 ⫾ 11.6a

aP

⬍ 0.01 compared with uninfected controls.

1.73a

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ically active, zonulin-containing media (36.9 ng/mg of protein) were tested on monkey small intestines mounted in Ussing chambers at a final concentration of 1 ng/mL. Compared with the amount of zonulin in the culture media, the final concentration of zonulin in the Ussing chamber’s Ringer’s solution represented a 1:10 dilution. Zonulin-containing cultures induced a time-dependent TEER decrease both in monkey jejunum (data not shown) and ileum (Figure 5) that was readily reversible on withdrawal of zonulin from the reservoir (data not shown). The permeating effect was completely abolished when tissues were preincubated with the zonulin inhibitor FZI/0 (1 ␮g/mL) (P ⬍ 0.05) (Figure 5), but not with the scrambled peptide FZI/1 (1 ␮g/mL)19 (data not shown), confirming that zonulin was responsible for the observed TEER decrement. No significant TEER changes were detected when zonulin was tested on monkey large intestine (data not shown), in keeping with the lack of zonulin receptor in the colon.10 Intestinal Cell Culture Experiments

Figure 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 (䊐). 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 ␮g/mL) completely prevented the inulin passage (■), whereas the scrambled peptide FZI/1 (1 ␮g/mL) (d) 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 bacteriaexposed small-intestinal tissues was not affected by preincubation with FZI/0 (E), 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.

Ussing Chamber Studies To establish whether the zonulin released in bacteria-exposed small-intestinal organ cultures was biolog-

Caco2 cells23 exposed to E. coli 6.1 secreted minimal amounts of zonulin in culture supernatants (0.67 ⫾ 0.06 ng/mg of protein per square millimeter). This secretion was paralleled by a TEER decrement of the cell monolayer (from 202.5 ⫾ 5.3 ⍀/cm2 to 170.1 ⫾ 6.8 ⍀/cm2; P ⬍ 0.05) that was prevented by pretreatment with the zonulin inhibitor FZI/0 (1 ␮g/mL) (from 201.5 ⫾ 11.9 ⍀/cm2 to 191.5 ⫾ 7.1 ⍀/cm2; P ⫽ not significant). No significant changes in either zonulin

Figure 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 (E) completely abolished the permeating effect that became indistinguishable from the negative control (}). The average baseline TEER was 42.5 ⍀/cm2; n ⫽ 4 observations.

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Figure 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 ␮g/mL) completely prevented the ZO1 redistribution (C and F ), suggesting that ZO1 disengagement from the junctional complex is zonulin dependent.

secretion as well as TEER changes were observed in uninfected Caco2 monolayers (data not shown). Interestingly, the same microorganism induced a 100-fold higher zonulin secretion when incubated with whole monkey small intestine (either jejunum, 53.4 ⫾ 8.6 ng/mg of protein per square millimeter, or ileum, 60.56 ⫾ 9.6 ng/mg of protein per square millimeter). Although at the moment we have no definitive explanation for the difference in zonulin secretion between Caco2 and whole intestinal tissue, several hypotheses can be formulated. Intestinal cells other than enterocytes may represent the main source of zonulin secretion after bacterial exposure; zonulin secretion from enterocytes could be up-regulated by extraepithelial cells; or human colon adenocarcinoma-derived cells (Caco2) and epithelial cultured cells in general may be less efficient at responding to bacteria or in secreting zonulin. Cell Culture Assays for ZO1 Localization by Direct Immunofluorescence Microscopy Several proteins that dictate paracellular permeability have been identified in the cytoplasmic submembranous plaque underlying intercellular tight junctions.

The better characterized component of this junctional complex is ZO1, a subcortical protein of approximately 225 kilodaltons whose C-terminus is functionally linked to the cell cytoskeleton, whereas its N-terminus is associated with the tight-junction protein occludin.24 Immunofluorescence analysis of human intestinal cell lines, Caco2 cells, and zonulin-sensitive rat intestinal cell monolayers (IEC6)11 was used to establish whether the zonulin release after bacterial colonization could affect the localization and/or migration of ZO1. Uninfected Caco2 monolayers showed the typical ZO1 localization at the cell periphery both after 30 minutes (Figure 6A) and 4 hours (Figure 6D) of incubation. Conversely, infected monolayers showed a redistribution of the ZO1 protein, with decreased staining at the edge of the cells (Figure 6B and E). These changes, typical of tight-junction disassembly,25 were already detected after 30 minutes of incubation (Figure 6B) and remained present after 4 hours of incubation (Figure 6E). Preincubation with FZI/0 (1 ␮g/mL) completely prevented the ZO1 redistribution in colonized monolayers (Figure 6C and F ), suggesting that the ZO1 disengagement from the junc-

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tional complex in bacteria-exposed monolayers is zonulin dependent. Similar results were obtained in IEC6 cells (data not shown).

Discussion Studies on the interaction between enteric bacteria and the intestinal host have mainly focused on the bacteria’s pathogenic mechanisms.26 In this study, we provided evidence that the small intestine may play an active, primary role in this enteric bacteria– host interaction by responding to the presence of microorganisms with the luminal secretion of zonulin. Our results showed that mammalian small intestines react to the exposure to either pathogenic or nonpathogenic enteric bacteria by activating the zonulin pathway, a system involved in the regulation of the small-intestinal tightjunction permeability.19 In the absence of enteric infections, the mammalian proximal small intestine is not colonized by bacteria. The colonization of the proximal gut by enteric microorganisms (even without apparent mucosal damage or elaboration of specific toxins) may lead to a leaky intestine.27 However, the mechanism by which this disturbed physiological regulation of the intestinal tight-junction permeability secondary to proximal bacterial contamination occurs remains unclear. Our results provide evidence that both normal enteric bacterial isolates (well characterized for not harboring any known pathogenic traits) and pathogenic bacteria induce alteration of the tight-junction competence, as suggested by changes in intestinal epithelial resistance and increased passage of inulin. These changes were mirrored by the concomitant expression of zonulin in organ culture systems. These results suggest that the presence of enteric microorganisms in the proximal small intestine (but not in the colon, where the zonulin system is not operative10) induces an intestinal mucosal response that leads to the luminal secretion of zonulin, probably as part of the host innate immune response. This hypothesis is further sustained by the following observations: (1) zonulin is not produced by the enteric microorganisms studied, because they did not harbor the zot gene or secrete Zot/zonulin antibody-reacting proteins in bacteria culture supernatants; (2) zonulin was secreted only when the bacteria were added to the luminal, but not the serosal, side of the small intestine; (3) this response was consistently elicited, irrespective of the mammalian species (rat, rabbit, or monkey) and microorganisms (virulent or avirulent) tested; and (4) the impairment of the intestinal barrier functions was blocked by pretreatment with the zonulin receptor binding inhibitor FZI/0. The fact that the interaction of bacteria with the small-intestinal mu-

GASTROENTEROLOGY Vol. 123, No. 5

cosa induces zonulin release can be interpreted as a mechanism of defense of the host that reacts to the abnormal presence of microorganisms and/or their cellwall components on the surface of the proximal small intestine. After the zonulin-induced opening of tight junctions, water is secreted into the intestinal lumen following hydrostatic pressure gradients,10 and bacteria are flushed out. Given the complexity of both the cell signaling events and the intracellular structures involved in the zonulin system,28 it is not surprising that this pathway may be affected when the physiological state of epithelial cells is continuously altered by proximal bowel contamination. The intestinal barrier dysfunction initiated by bacterial infections has been hypothesized as the main pathogenic factor in several pathologic conditions involving the gastrointestinal tract.29 There now seem to be a spectrum of diseases that are associated with an aberrant intestinal presentation of environmental antigens. The increased tight-junction permeability seems to be a common denominator in these diseases and may be responsible for the repeated passage of luminal antigens to the mucosal immune system. The response to this passage is dictated by the genetic susceptibility of the host immune system to recognize, and potentially misinterpret, an environmental antigen presented within the gastrointestinal tract. As a consequence, either allergic reactions to food allergens or autoimmune diseases may develop.12 Our recent observation that increased zonulin expression and impairment of the gut barrier function are common findings of the early phase of celiac disease9 and precede the onset of diabetes in a rat model of type 1 diabetes mellitus30 further supports the hypothesis that the abnormal production of this protein, possibly secondary to proximal bacteria contamination, is involved in the pathogenesis of autoimmune disorders such as diabetes mellitus and celiac disease.

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Received September 25, 2001. Accepted August 1, 2002. Address requests for reprints to: Alessio Fasano, M.D., Division of Pediatric Gastroenterology and Nutrition, University of Maryland School of Medicine, 685 W. Baltimore Street, HSF Building, Room 465, Baltimore, Maryland 21201. e-mail: [email protected]; fax: (410) 328-1072. This article was partially supported by National Institutes of Health grant DK-48373 (to A.F.) and by a grant of the Italian Society of Pediatric Gastroenterology and Hepatology (to R.E.A.).