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From the Department of Pathology and Molecular Medicine, Intestinal Disease Research Programme, McMaster University, Hamilton, Ontario, Canada
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Abstract |
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Under normal circumstances the epithelial lining of the gut forms a semipermeable barrier, the selectivity of which is in large part determined by the intercellular tight junctions. The tight junctions consist of interdigitating occludin molecules and one or more claudins that are linked to the actin cytoskeleton via a complex of proteins including the zonula occludens (ZO) 1, 2, and 3.4,5
Recent evidence suggests that members of the claudin family play a critical role in tight junction formation and determine the permeability characteristics in a variety of tissues including the gut.6
Claudin-1 in particular is believed to regulate epithelial permeability. Evidence includes the lethality of claudin-1-deficient mice,7
reduced claudin-1 expression after interferon (IFN)-
-induced barrier defects in thyrocytes,8
increased claudin-1 expression leading to barrier enhancement of airways and kidney epithelia,9,10
and the significant redistribution of claudin-1 at the onset of rotavirus-induced paracellular permeability in intestinal epithelial monolayers.11
Investigations with model epithelial cell lines have shown that EHEC forms intimate attaching and effacing lesions on host cells by eliciting and modulating a variety of intracellular signaling pathways to reorganize the cytoskeleton while evoking a significant increase in epithelial permeability. This diminished epithelia barrier is accompanied by loss of ZO-1 from the tight junction region12 and may be of pathological significance by allowing pathogens and antigens unimpeded access to the mucosa. Indeed, the paracellular pathway may be one way for Shiga toxin to enter the circulation because the receptor for the toxin has not been identified on human gut epithelium.13 Thus, agents capable of enhancing or maintaining the epithelial barrier, through preservation of the tight junction or via modulating intracellular signaling pathways that compete with those activated by bacteria, may be of value in reversing the effects of EHEC infection.
Transforming growth factor (TGF)-ß is a multifunctional cytokine with immunosuppressive properties produced by many cell types including intestinal epithelia and can enhance epithelial barrier function. For instance, monolayers of human colon-derived epithelia treated with TGF-ß display an increase in transepithelial electrical resistance (TER).14
Moreover, TGF-ß also prevents or reduces epithelial barrier disruption caused by infection with Cryptosporidium parvum, exposure to IFN-, or exposure to conditioned medium from activated immune cells.14-17
However, neither the ability of TGF-ß to ameliorate EHEC-induced barrier disruption nor the mechanisms underlying preservation of epithelial barrier function have been reported. Thus, the aims of the present study were 1) to define the mechanism behind TGF-ß enhancement of epithelial barrier function in terms of intracellular signaling pathways and tight junction protein expression, 2) to characterize the effects of EHEC on tight junction protein expression and distribution, and 3) to determine whether TGF-ß could block the EHEC-induced increase in epithelial permeability.
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Materials and Methods |
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T84 cells were grown and maintained in a 1:1 (vol/vol) mixture of Dulbecco’s modified Eagle’s medium and Ham’s F-12 medium, supplemented with 10% fetal bovine serum, 1.5% HEPES, and 2% penicillin-streptomycin (all from Life Technologies, Grand Island, NY) at 37°C, 5% CO2. Enterohemorrhagic E. coli (EHEC) O157:H7 (strain CL-56)12 and enteropathogenic E. coli (EPEC) O127:H6 (strain E2348/69)18 were maintained on 5% sheep blood agar plates (PML Microbiologicals, Mississauga, Canada) at 4°C, cultured overnight in Luria-Bertani broth at 37°C with continuous agitation, diluted in Luria-Bertani broth, and then grown for 4 to 6 hours (to mid-logarithmic growth phase) before T84 monolayer infection. The bacterial suspension was pelleted (2500 rpm, 20 minutes), resuspended in antibiotic-free T84 cell culture medium, and 108 CFU were added to the apical surface of epithelial monolayers at an MOI of 100 (multiplicity of infection; 100 infectious units/epithelial cell). A laboratory strain of nonpathogenic E. coli, HB101, was used as a control and was cultured as described above.19
Measurement of Epithelial Barrier
T84 cells were seeded onto filter supports (106 cells/well, surface area 1 cm2; Costar Inc., New York, NY) and grown to confluence (
6 days), at which point TER was monitored by a voltmeter and chopstick electrodes (Millicell-ERS; Millipore, Bedford, MA). To assess the effects of TGF-ß, the cytokine was added to the basolateral surface of T84 monolayers (human recombinant TGF-ß1, 10 ng/ml; R&D Systems, Minneapolis, MN), and TER was recorded at 4, 8, 16, 24, 48, and 72 hours after treatment. Based on previous studies showing that TGF-ß has barrier-enhancing effects at doses of 1 to 100 ng/ml,14,20
we selected a dose of TGF-ß of 10 ng/ml for this study. The acute effects of TGF-ß were assessed by wash out experiments, in which monolayers were exposed to TGF-ß for 1 hour, rinsed twice with culture media, and maintained daily in fresh culture media with TER recorded at 24-hour intervals. For pharmacological inhibition of ERK MAPK, T84 monolayers were pretreated with the MEK1 inhibitor PD98059 (apical and basolateral, 1 hour, 25 µmol/L; Calbiochem, San Diego, CA20
), exposed to TGF-ß (10 ng/ml, 1 hour), rinsed, and maintained with fresh culture media for 72 hours, and TER was then recorded.
In bacterial co-culture experiments, T84 monolayers were treated with TGF-ß (10 ng/ml) 45 minutes before infection, with TGF-ß remaining for the duration of infection, and TER recorded 16 hours later (a time point at which EHEC infection reduces epithelial barrier function12
). In additional studies, 2.5 µCi of [3H]-mannitol (
180 d; Sigma Chemical Co., St. Louis, MO) was added to the apical side of filter-grown monolayers at the end of the experiment. As an inert small molecule, mannitol will cross the epithelium predominantly via the paracellular pathway. Four hours later 10-µl and 500-µl aliquots of culture medium were obtained from the apical and basal culture well, respectively, and radioactivity determined in a scintillation counter (Becton Dickinson, Mississauga, Canada). The transepithelial passage of the labeled mannitol was calculated.
Adenoviral Infection
T84 cells were seeded as described above and, at 4 hours after seeding, infected for 16 hours with replication-deficient adenovirus constructs encoding SMAD7 (Ad-SMAD7, MOI = 50; a generous gift from Dr. ten Dijke, The Netherlands Cancer Institute, Leiden, The Netherlands).21,22 Cells were rinsed, grown for 3 days, and then treated with TGF-ß (10 ng/ml, 24 hours) and TER recorded 72 hours after cytokine exposure.
Immunoprecipitation for SMAD2/3
T84 monolayers were seeded onto filter supports (3 x 106 cells/well, surface area 4.7 cm2; Costar Inc.), grown to confluence, serum-starved for 24 hours, and treated with TGF-ß (10 ng/ml, 5 to 60 minutes). Whole cell protein extracts were prepared, protein concentrations determined,23 and samples (1 mg/ml in phosphate-buffered saline) incubated with gentle agitation at room temperature for 2 hours with mouse anti-human SMAD2/3 antibody (2 µg/ml; Santa Cruz Biotechnology, Santa Cruz, CA), followed by centrifugation and incubation with protein A-conjugated beads (Easy-View; Sigma-Aldrich, St. Louis, MO). Immunoprecipitated SMAD2/3 was recovered following the manufacturer’s protocol and loaded immediately onto a 10% sodium dodecyl sulfate-polyacrylamide gel electrophoresis gel.
Immunoblotting
Samples were prepared from T84 monolayers as described above ± TGF-ß (10 ng/ml) ± bacteria (MOI = 100). Proteins (40 µg/sample) were electroblotted onto a nitrocellulose membrane (Bio-Rad Laboratories, Hercules, CA) and blocked in 5% bovine serum albumin in Tris-buffered saline (containing 0.1% Tween-20, TBST) or 5% milk diluted in TBST, according to the manufacturer’s directions.
Phospho-SMAD2/3, SMAD2/3, and SMAD7
Blots were probed with mouse anti-human phospho-serine antibody (phospho-SMAD2/3), rabbit anti-human SMAD2/3, or goat anti-human SMAD7 diluted in bovine serum albumin-TBST (1:1000, 1:1000, and 1:750, respectively; 1 hour at room temperature; Abcam, Cambridge, UK), washed in TBST, and exposed to the appropriate horseradish peroxidase-conjugated secondary antibodies (1:3000, Santa Cruz Biotechnology).
FLAG
Blots were probed with mouse anti-FLAG diluted in milk-TBST for 1 hour at room temperature (1:1000, Sigma-Aldrich), washed in TBST, and exposed to horse-radish peroxidase-conjugated rabbit anti-mouse IgG (1:3000, 1 hour room temperature; Santa Cruz Biotechnology).
Phospho-ERK1/2 and Tight Junction Proteins
Blots were either probed for 1 hour at room temperature (mouse anti-human phospho-ERK1/2, 1:800; rabbit anti-human ERK1/2, 1:3000; Santa Cruz Biotechnology) or overnight at 4°C (rabbit anti-human claudin-1, 0.5 µg/ml; rabbit anti-human claudin-2, 1.0 µg/ml; mouse anti-human claudin-4, 1.5 µg/ml; rabbit anti-human occludin, 0.25 µg/ml, rabbit anti-human ZO-1, 1.5 µg/ml; Zymed Laboratories, San Francisco, CA), washed in TBST (5x) and then exposed to the appropriate horseradish peroxidase-conjugated secondary antibodies (1:2000 to 1:15,000, 1 hour room temperature; Santa Cruz Biotechnology). Immunoreactive proteins were visualized using chemiluminescence (ECL; Amersham Pharmacia, Piscataway, NJ) and exposure to Kodak XBL film (Eastman Kodak Co., Rochester, NY). Initial tight junction protein immunoblots were stripped and reprobed for ß-actin (goat anti-human actin, 1:200; bovine anti-goat 1:400; Santa Cruz Biotechnology) to assess total protein loading. Subsequently, we used claudin-4 expression as an internal control because this did not change with infection or TGF-ß treatment and is a tight junction protein. The densitometry ratio between the tight junction protein of interest and the internal control from each extraction was compared between control (ie, naïve) and treated epithelia.
Confocal Microscopy
T84 cells were seeded onto filter supports (105 cells/well, surface area 0.33 cm2) and either treated with TGF-ß for 72 hours (10 ng/ml) or infected with EHEC O157:H7 for 16 hours (MOI = 100) ± TGF-ß pretreatment for 45 minutes (10 ng/ml) (as described above). Monolayers were rinsed in phosphate-buffered saline, fixed in 4% formalin, permeabilized with 0.1% Triton X-100 (Mallinckrodt Inc., Paris, KY), and blocked for 30 minutes in blocking solution [5% bovine serum albumin/5% goat serum (Sigma-Aldrich) in 0.1% Triton X-100]. Primary and secondary antibodies were diluted in blocking solution (rabbit anti-human claudin-1, 15 µg/ml, mouse anti-human claudin-2, 2 µg/ml, or rabbit anti-human ZO-1, 2 µg/ml, Zymed Laboratories; Alexa Fluor 488 goat anti-rabbit IgG or Alexa Fluor 488 goat anti-mouse IgG, 1.5 µg/ml, Molecular Probes, Eugene, OR) and exposed to monolayers for 1 hour at room temperature. After extensive washing, filters were excised from the polystyrene filter supports, and mounted on glass slides in Gel/Mount anti-fade reagent (Biomeda Corp., Foster City, CA). Images were acquired using an inverted laser-scanning microscope (LSM 510, Axiovert 100 mol/L; Zeiss, Oberkochen, Germany) equipped with argon (450 to 514 nm). Immunoreactive claudin and ZO-1 were excited using the 488-nm laser and collected using a standard fluorescein isothiocyanate filter set. For each experiment, image acquisition (ie, confocal microscope settings) and processing was identical between controls and TGF-ß-, EHEC-, or TGF-ß + EHEC-treated cells. Images are presented as either en face to illustrate the distribution of the tight junction protein immunoreactivity or as a composite Z-stack reconstruction, which shows the monolayer in transverse profile with the basally located nuclei identified by propidium iodide staining (red) and the tight junction proteins by fluorescein isothiocyanate-labeled secondary antibodies (green).
[3H]-Thymidine Incorporation
T84 cells were seeded onto 96-well plates (4 x 104 cells/well) and grown for 4 days. [3H]-Thymidine (1 µCi/well; DuPont-New England Nuclear, Wilmington, DE) was added for the duration of TGF-ß treatment (10 ng/ml, 72 hours). Cells were harvested onto glass-fiber filter disks and assayed in 5 ml of aqueous counting scintillant for ß-ray emission, and counts per minute were determined in a scintillation counter (Becton Dickinson). Results are expressed as mean cpm ± SEM.
Bacterial Growth
EHEC was cultured to mid-logarithmic growth phase (described in Cell and Bacterial Culture), resuspended in antibiotic-free T84 cell culture media ± TGF-ß (10 ng/ml), and incubated at 37°C with continuous agitation for 16 hours. Bacterial growth was determined by spectrophotometric analysis of each sample (OD600: 0.1 = 109 CFU). In similar experiments, supernatants from naïve T84 monolayers or TGF-ß-treated T84 cells were added to EHEC in mid-logarithmic growth phase, cultured as above, and OD600 measured.
Interleukin (IL)-8 Enzyme-Linked Immunosorbent Assay
T84 cells were seeded onto filter supports (106 cells/well, surface area 1 cm2), grown to confluence, and infected with EHEC ± TGF-ß pretreatment (as described above) for 16 hours. Subsequently, culture medium samples were collected from the basolateral wells and frozen at –20°C until the concentration of IL-8 was determined by commercial enzyme-linked immunosorbent assay (R&D Systems). Detection limit of the assay was 16 pg/ml.
Statistical Analysis
Data are presented as means ± SEM. Data were analyzed using one-way analysis of variance followed by posthoc comparisons using Tukey’s test, or Student’s t-test, with P < 0.05 accepted as the level of statistical significance.
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Results |
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Figure 1
shows that TGF-ß (10 ng/ml) evoked a time-dependent increase in TER that was significant at 16 hours after treatment and more pronounced at 72 hours when there was a twofold increase greater than pretreatment values. Epithelial TER was similarly increased 72 hours after a single 1-hour pulse with TGF-ß, indicating that acute exposure to this cytokine has long-term effects (Figure 1
, bar labeled 72h
). Indeed, our earlier publication showing that 1-hour TGF-ß treatment causes alterations in epithelial ion transport 16 hours later supports this observation.20
Reflecting the high TER baseline of control monolayers, there was no significant difference in [3H]-mannitol flux across control and TGF-ß-treated epithelial monolayers.
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is an immunoblot showing ERK 1/2 activation (ie, increased phosphorylation) in whole cell protein extracts of T84 cells after 15 and 30 minutes of TGF-ß exposure. Figure 2B
shows increased levels of phosphorylated SMAD2/3 30 to 60 minutes after cytokine treatment, indicating SMAD2/3 activation. SMAD7 is a naturally occurring inhibitor of the SMAD signaling pathway that is typically induced after TGF-ß activation and provides a negative feedback mechanism for TGF-ß signaling.24
In TGF-ß-treated T84 epithelia, SMAD7 expression was increased 4 hours after cytokine exposure and remained elevated at 24 hours (end of experiment, Figure 2C
). Inhibition of ERK MAPK and SMAD signaling via the pharmacological inhibitor PD98059 and via Ad-SMAD7, respectively, completely prevented the TGF-ß-induced increase in epithelial TER (Figure 3)
.
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To determine the structural mechanism behind the epithelial barrier enhancement, we compared tight junction protein expression between TGF-ß-treated T84 monolayers and untreated controls. TGF-ß exposure (10, 50, or 100 ng/ml, 72 hours) resulted in twofold to threefold up-regulation of claudin-1, whereas neither claudin-2 nor claudin-4 were significantly affected (Figure 4)
. Figure 4B
(i) shows densitometry analysis of the representative blot shown in Figure 4A
, and Figure 4B
(ii) shows quantification of the increased claudin expression in six epithelial preparations exposed to TGF-ß (10 ng/ml, 72 hours) compared to whole cell protein extracts from time-matched controls. Supporting the TER data (Figure 3)
, inhibition of ERK MAPK and SMAD signaling reduced the TGF-ß-induced increases in claudin-1 expression (Figure 4C)
. The increased claudin-1 could have been retained solely in a central cytosolic position. However, confocal microscopy revealed a chicken-wire pattern of claudin-1 immunoreactivity that is typical of tight junction protein expression at the margins of polarized epithelia (Figure 4D)
. Moreover, assessment of z axis reconstructions showed that the claudin-1 immunoreactivity was similar in control and TGF-ß-treated monolayers and was mostly confined to the apical lateral membranes. However, there was a tendency for claudin-1 to extend further down the lateral membrane in epithelia exposed to TGF-ß (immunostaining was noted in the cytosolic compartment in both treated and control epithelial preparations, consistent with normal protein production and turnover). Neither occludin nor ZO-1 expression were consistently or significantly affected by TGF-ß treatment (data not shown). The increased claudin-1 expression was not simply a reflection of increased proliferation because [3H]-thymidine incorporation was actually reduced after 72 hours of TGF-ß exposure (31,477 ± 4861 versus 5294 ± 612 cpm, control versus TGF-ß respectively, n = 12; P < 0.001).
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TGF-ß protects T84 monolayers against loss of barrier function caused by infection with C. parvum.15
Thus, we tested whether TGF-ß (10 ng/ml) could prevent the barrier dysfunction caused by other pathogenic bacteria by challenging TGF-ß-treated T84 monolayers with EHEC O157:H7 (MOI = 100, 16 hours). Figure 5
shows that a 45-minute TGF-ß pretreatment prevented the decrease in TER caused by EHEC infection. EHEC infection caused an 30-fold increase in transepithelial flux of [3H]-mannitol: control = 0.49 ± 0.13% versus EHEC infection = 10.63 ± 2.36% of apical mannitol (based on cpm; n = 10 T84 monolayers from four experiments; P < 0.05). TGF-ß pretreatment of the epithelial monolayer reduced the EHEC barrier disruption as gauged by [3H]-mannitol flux (control = 0.79 ± 0.09, EHEC = 21.34 ± 3.42, TGF-ß + EHEC = 11.61 ± 2.51% of apical mannitol; n = 3 monolayers from one of two experiments).
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EHEC Infection Causes Reduced Tight Junction Protein Expression
Infection with pathogenic bacteria, such as EHEC, EPEC, and Shigella flexneri, decreases expression of tight junctions and associated proteins.12,25-28
However, little is known about their effects on claudin protein expression. Figure 6A
shows representative immunoblots from an analysis of tight junction proteins in our model system. Corroborating our earlier immunolocalization studies,12
ZO-1 expression was reduced by 34 ± 3% 16 hours after EHEC infection (n = 9 T84 cell extracts from three experiments). Similarly, occludin expression was reduced by 27 ± 5% by EHEC infection compared to controls (n = 6 T84 cell extracts from two experiments). Of the three claudins examined, only claudin-2 was significantly reduced by EHEC: claudin-2 expression was significantly reduced by 16 ± 2%* compared to controls (n = 15 T84 cell extracts from five experiments, *P = 0.03; Figure 6B
).
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. Furthermore, confocal microscopy revealed an obvious disruption of the normal distribution of claudin-2-like immunoreactivity in EHEC-treated T84 cell monolayers, an effect that was virtually completely prevented if the cells were pretreated with TGF-ß (10 ng/ml, 45 minutes) (Figure 7C)
. In addition, the accompanying Z-stack compilation images in Figure 7C
(each one below the appropriately labeled en face image) not only confirm the lack of claudin-2 expression in EHEC-treated epithelia, they also demonstrate that although claudin-2 can be found throughout the cell, there is clear accumulation of the immunoreactivity at the cell’s apical pole and juxtaposed to a location compatible with the tight junction. The distribution of claudin-2 immunoreactivity in control cells (Ci) and in TGF-ß + EHEC (TEi)-treated T84 cell monolayers was virtually indistinguishable (Figure 7C)
. Finally, confocal microscopic assessment revealed that epithelial monolayers treated with TGF-ß displayed ZO-1 immunoreactivity patterns comparable to controls. In contrast, EHEC-infected T84 cells (MOI = 100, 16 hours) had strikingly reduced ZO-1 protein expression (matching the immunoblot analysis). TGF-ß pretreatment (45 minutes, 10 ng/ml) prevented this EHEC-induced rearrangement of ZO-1 (Figure 7D)
.
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Because cytokines can possess antimicrobial activity,25 we tested whether TGF-ß itself, or epithelial factors induced by TGF-ß (eg, antimicrobials), could impair EHEC growth. TGF-ß did not directly affect EHEC viability after 16 hours of co-incubation (OD600: EHEC 1.3 ± 0.1 versus TGF-ß + EHEC 1.3 ± 0.1; n = 2). Bacterial growth was similarly unaffected by supernatants from TGF-ß-treated T84 monolayers (data not shown), suggesting that an epithelial-derived factor was also not involved in the preservation of epithelial barrier function via induction of bacterial cell death or inhibition of bacterial proliferation.
TGF-ß Does Not Prevent EHEC-Induced IL-8 Production by Intestinal Epithelia
EHEC infection induces IL-8 production from intestinal epithelia.29 Given the protective effects of TGF-ß on barrier function, we tested whether pretreatment with TGF-ß would prevent EHEC-induced IL-8 production from T84 monolayers. In comparison to untreated control monolayers or TGF-ß-treated epithelia (which produced 108 ± 18 pg/ml and 130 ± 20 pg/ml of IL-8, respectively), EHEC-infected T84 cells produced significant amounts of IL-8 that was not prevented by TGF-ß pretreatment (193 ± 27* pg/ml and 186 ± 27* pg/ml, respectively; *P < 0.05 compared to control and TGF-ß only; n = 5 to 6 monolayers).
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Discussion |
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TopAbstractMaterials and MethodsResultsDiscussionReferences |
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This study corroborates our earlier findings20
and those of others,16
showing that TGF-ß enhances the epithelial barrier (at least as gauged by TER). Subsequent molecular assessment of TGF-ß-treated T84 epithelia revealed an expected increase in ERK MAPK and SMAD2/3 activation as judged by phosphorylation on immunoblots. Moreover, use of inhibitors of ERK and SMAD2/3 signaling blocked TGF-ß-enhancement of barrier function. Whether these pathways regulate epithelial paracellular permeability independently or via intracellular cross-talk (Figure 8)
31
remains to be determined. TER, although primarily reflective of tight junction permeability, can also be influenced by membrane ion channels and transporter activity. We found that TGF-ß reduced expression of a Cl– channel (ie, the CFTR).32
However, unlike the present study the ion transport effect was insensitive to ERK blockade with PD98058,20
suggesting that the TGF-ß-induced increased TER is due to modulation of the tight junction. Also noteworthy, is the fact that a 1-hour TGF-ß pulse resulted in increased TER 72 hours later. This lasting effect of TGF-ß on epithelial barrier enhancement is unlike the effects of other cytokines, for example IFN-, in which the cytokine-induced barrier defect is not observed with an exposure of less than 6 hours duration (D. M. McKay, unpublished observation).
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In addition to barrier enhancement, TGF-ß preserves intestinal epithelial barrier function after exposure to agents known to cause barrier disruption, such as IFN-, supernatants from superantigen-stimulated immune cells, and infection with C. parvum.14,15,17
Here we show that TGF-ß protects against intestinal epithelial barrier dysfunction caused by infection with EHEC (and EPEC), which is not due to effects on EHEC growth or viability. EHEC and EPEC infections are associated with tight junction changes. Specifically, EHEC infection disrupts the distribution of ZO-1,12
and EPEC causes dephosphorylation of occludin and its subsequent dissociation from the tight junction.26
We observed that pretreatment with TGF-ß prevented the EHEC-induced reductions in occludin and ZO-1 and the disruption of ZO-1 distribution. Also, considering the important role of claudin proteins in tight junction formation35
and the claudin-4 epithelial barrier disruption caused by Clostridium perfringens,36
we examined the effects of EHEC infection on claudin expression. Our data show that in the colon-derived T84 cell line, EHEC specifically affects claudin-2 but not claudin-1 or claudin-4, as exhibited by the significant reduction in claudin-2 protein expression and dissociation from the tight junction (preliminary data suggest that EPEC similarly affects claudin-2 expression; unpublished observation). The EHEC-induced reduction in claudin-2 expression was significantly less pronounced in TGF-ß-treated cells, correlating with the preservation of the other tight junction proteins (ie, occludin, ZO-1) and the maintenance of the epithelial barrier (ie, TER, [3H]-mannitol flux). At present, the exact role of claudin-2 in tight junction barrier function is unclear. Furuse and colleagues,37
showed that introduction of claudin-2 into high-resistance Madin-Darby canine kidney (MDCK) epithelia caused decreased TER and led to the concept that claudin-2 forms conductive pores within tight junction strands. Indeed, claudin-2 has since been shown to induce cation-selective channels in tight junctions in the MDCK cell line.38
In contrast, induction of claudin-2 expression in low-resistance MDCK cells led to a 20% increase in TER.39
Furthermore, using T84 cells monolayers Nishiyama and colleagues40
found that increased TER was associated with up-regulation of claudin-2 protein expression at the tight junction. Similarly, the loss of barrier integrity observed in IFN- + tumor necrosis factor-
-treated T84 cell monolayers was accompanied by reduced claudin-2 expression,41
and, conversely, the enhancement of the barrier property of Caco-2 epithelial cells (another human colon-derived cell line) by retinoic acid was associated with increased claudin-2 expression.42
Thus, the exact role of claudin-2 in epithelial barrier function is controversial, may be cell-type-specific, and may be influenced by the other constituents of the tight junction. Collectively our data suggest that the structural basis for barrier preservation by TGF-ß lies in preventing EHEC-mediated down-regulation of claudin-2, occludin, and ZO-1 protein expression and in maintaining the normal distribution of these proteins. Because we found no evidence of direct TGF-ß up-regulation of claudin-2 expression, we speculate that TGF-ß antagonism of EHEC-induced disruption of epithelial barrier function could be due to interference with a bacteria-induced epithelial signaling pathway43
or stabilization of claudin-2 mRNA (Figure 8)
.
In terms of other effects on host cell function by EHEC, Dahan and colleagues44
determined that S. boulardii significantly decreased epithelial IL-8 secretion during EHEC infection by inhibiting the MAPK and nuclear factor (NF)-
B signaling pathways responsible for this proinflammatory response. Given the anti-inflammatory properties of TGF-ß, we tested the ability of TGF-ß to prevent epithelial IL-8 secretion during EHEC infection. TGF-ß did not prevent EHEC-induced epithelial IL-8 secretion, indicating that the effect of TGF-ß was specific for protection against EHEC-mediated disruption of the epithelial barrier. However, we cannot exclude the possibility that TGF-ß may regulate other aspects of an EHEC-induced proinflammatory response. For example, TGF-ß exerts anti-inflammatory properties against Bacteroides vulgatus- and lipopolysaccharide-mediated NF-B activation and subsequent IL-6 secretion in intestinal epithelia.45
Because activation of NF-B regulates not only IL-8 gene expression but also the expression of several other proinflammatory mediators such as tumor necrosis factor- and IL-6,46,47
TGF-ß may exert anti-inflammatory properties against EHEC-induced expression of such genes. Finally, EHEC-derived Shiga toxin and its translocation across the epithelium pose a significant biological threat to renal function that may culminate in hemolytic uremic syndrome. It is therefore noteworthy that elevated levels of TGF-ß are associated with reduced incidence of hemolytic uremic syndrome in children infected with EHEC O157:H7,48
making it feasible that increased TGF-ß enhances epithelial barrier function and limits toxin entry into the body.
TGF-ß influences many aspects of intestinal epithelial homeostasis. We found that TGF-ß enhancement of epithelial barrier function was associated with increased claudin-1 expression, an ERK MAPK- and SMAD2/3-dependent event. In addition, preservation of epithelial barrier integrity in the face of EHEC infection by TGF-ß was accompanied by maintenance of the levels and distribution of claudin-2, occludin, and ZO-1 (Figure 8)
. Thus, awareness of TGF-ß enhancement of epithelial barrier function is now complemented by data on the intracellular signaling pathways and structural modification of the tight junctions that mediate this physiological and protective effect of TGF-ß in the intestine. Overall, the data presented offer compelling evidence that differential regulation of tight junction protein expression, particularly claudins, by TGF-ß provides beneficial effects capable of enhancing the epithelial barrier and protecting it against severe insult by pathogenic bacteria.
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Acknowledgements |
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Footnotes |
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Supported by the Canadian Institutes of Health Research (grant MT-13421 to D.M.M.).
K.L.H. has been a recipient of a Natural Sciences and Engineering Research Council studentship (2001 to 2003) and a Canadian Institutes of Health Research/Canadian Digestive Health Foundation partnership award (2003 to 2004).
Accepted for publication August 11, 2005.
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References |
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TopAbstractMaterials and MethodsResultsDiscussionReferences |
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down-regulates claudin-1 and impairs the epithelial barrier function in primary cultured human thyrocytes. Eur J Endocrinol 2003, 149:215-221[Abstract]
and tumor necrosis factor-. Inhibition of increased permeability, but not diminished secretory responses by transforming growth factor ß2. J Immunol 1997, 159:2382-2390B-independent pathway. FASEB J 2003, 17:1319-1321B and AP-1 in T84 cells. Infect Immunity 2002, 70:2304-2310B recruitment to the interleukin-6 gene promoter in intestinal epithelial cells through modulation of histone acetylation. J Biol Chem 2003, 278:23851-23860B regulation in the immune system. Nat Rev Immunol 2002, 2:725-734[Medline]
and IL-6 production through NF-B in peritoneal mast cells. Biochim Biophys Acta 2003, 1643:75-83[Medline]
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·cm2 and 607 ± 158 
