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GASTROINTESTINAL, HEPATIC, PULMONARY, AND RENAL

Phosphatidylinositol 3'-Kinase Is a Critical Mediator of Interferon--Induced Increases in Enteric Epithelial Permeability

Intestinal Disease Research Programme (D.M.M., J.L.W., A.W., J.C., D.P., P.M.J.C., J.L.), Department of Pathology and Molecular Medicine, McMaster University, Hamilton, Ontario, Canada; and Department of Surgical and Gastroenterological Sciences, University Hospital Padova, Italy (V.D.L.)

Received September 8, 2006; accepted December 15, 2006.

	Abstract

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The epithelial lining of mucosal surfaces acts as a barrier to regulate the entry of antigen and pathogens. Nowhere is this function of the contiguous epithelium more important than in the gut, which is continually exposed to a huge antigenic load and, in the colon, an immense commensal microbiota. We assessed the intracellular signaling events that underlie interferon (IFN) -induced increases in epithelial permeability using monolayers of the human colonic T84 epithelial cell line. Confluent epithelial monolayers on semipermeable supports were treated with IFN (20 ng/ml), and barrier function was assessed 48 h later by measuring transepithelial electrical resistance (TER: reflects passive ion flux), fluxes of ⁵¹Cr-EDTA and horseradish peroxidase (HRP), and transcytosis of noninvasive, nonpathogenic Escherichia coli (strain HB101). Exposure to IFN decreased barrier function as assessed by all four markers. The phosphatidylinositol 3'-kinase (PI-3K) inhibitors, LY294002 [2-(4-morpholinyl)-8-phenyl-1(4H)-benzopyran-4-one hydrochloride] and wortmannin, did not affect baseline permeability characteristics but completely blocked the drop in TER, increased fluxes of ⁵¹Cr-EDTA and HRP, and significantly reduced E. coli transcytosis evoked by IFN. In addition, use of the pan-protein kinase C (PKC) inhibitor, bisindolylmaleimide I (5 µM), but not rottlerin (blocks PKC), partially ameliorated the drop in TER and inhibited increased E. coli transcytosis. Addition of the PI-3K and PKC inhibitors to epithelia 6 h after IFN exposure still prevented the increase in paracellular permeability but not E. coli transcytosis. Thus, IFN-induced increases in epithelial paracellular and transcellular permeability are critically dependent on PI-3K activity, which may represent an epithelial-specific target to treat immune-mediated loss of barrier function.

With respect to gut homeostasis and mucosal immunity, it is critical that the epithelium limits the access of potentially dangerous antigen and microbes into the mucosa, and, as such, it is not surprising that enteric disease is often accompanied by increased gut permeability (Söderholm et al., 2002). Under normal circumstances, lumen-derived material crosses the epithelium by either transcellular or paracellular routes: involving transit across both apical and basolateral membranes via host endocytic/exocytic processes (or active pathogen invasion) or permeation of the intercellular tight junction protein complexes, respectively. The epithelial barrier is not static, but it is regulated by exogenous stimuli (e.g., bacterial toxins) (Philpott et al., 1996) and endogenous factors (e.g., cytokines) (Prasad et al., 2005). Numerous examples of cytokine regulation of epithelial paracellular permeability and tight junction structure/function have accumulated. For instance, exposure of monolayers of human colon-derived epithelial cell lines to interferon (IFN) (± other cytokines, such as tumor necrosis factor-) results in a significant increase in monolayer permeability (Madara and Stafford, 1989; Ivanov et al., 2004; Watson et al., 2004; Wang et al., 2005). Although less extensive, in vivo studies have implicated IFN in increased gut permeability evoked by stress and inflammation, corroborating the in vitro analyses (Yang et al., 2002; Cenac et al., 2004).

IFN production is increased in a variety of diseases, and given its ability to affect epithelial integrity, we and others have pursued the mechanism by which IFN leads to increased epithelial permeability, with the goal of elucidating a specific target that when antagonized prevents this effect of IFN, maintaining the barrier property of the epithelium. Since the original observations of IFN-induced increases in epithelial function (Madara and Stafford, 1989), it has become clear that this is not due to the induction of apoptosis (Bruewer et al., 2003) but instead is associated with rearrangement of the actin cytoskeleton and internalization and reduced expression of tight junction proteins (Youakim and Ahdieh, 1999; Utech et al., 2005). However, less is known of events that transduce IFN-IFN receptor interaction into the physiological outcome of increased epithelial permeability, an event that takes 24 to 48 h to manifest and is dependent on protein synthesis. Moreover, there is a dearth of data on IFN effects on transcellular permeability, which has recently been presented as a portal of entry for bacteria into the gut mucosa (Nazli et al., 2004; Clark et al., 2005).

Recently, we showed that pharmacological blockade of IFN-induced signal transducer and activator of transcription (STAT)-1 activation did not prevent the IFN-induced reduction in transepithelial electrical resistance (an index of paracellular permeability) (Watson et al., 2004). Extending these observations, we present data in support of phosphatidylinositol 3'-kinase (PI-3K) and protein kinase C (PKC) as mediators of IFN-induced increases in epithelial paracellular permeability and the transcytosis of noninvasive, nonpathogenic Escherichia coli across monolayers of human gut-derived epithelial cells.

	Materials and Methods

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Cell Culture and Reagents
The human colon-derived T84 epithelial cell line was cultured at 37°C with 5% CO₂ in a 1:1 mixture of Dulbecco's modified Eagle's medium and Ham's F-12 medium supplemented with 2% (v/v) penicillin-streptomycin and 1.5% (v/v) HEPES (all from Invitrogen, Burlington, ON, Canada) and 10% fetal bovine serum (CanSera, Toronto, ON, Canada). Human recombinant IFN and interleukin (IL)-4 were from R&D Systems (Minneapolis, MN). The following reagents were purchased from Sigma Chemical Co. (St. Louis, MO): LY294002 and wortmannin (both inhibit PI-3K), (-)-epigallacatechin gallate (EGCG; green tea polyphenol that inhibits STAT1 phosphorylation), N-nitro-L-arginine methyl ester [L-NAME; inhibits nitric oxide synthase (NOS)], and bisindolylmaleimide I (BIM; inhibitor of PKC isoforms). The phosphatidylinositol analog (1-L-6-hyrdomethyl-chiro-inositol 2-[(R)-2-O-methyl-3-O-octadecylcarbonate]; blocks PH domain protein interactions), AKT inhibitor II [SH-5 and API-2 (triciribine)], rottlerin (blocks PKC), N^G-monomethyl-L-arginine (L-NMMA; inhibits NOS), and N⁶-(1-iminoethyl)-lysine HCl (L-NIL; inhibits inducible NOS) were from Calbiochem (San Diego, CA). Enteropathogenic E. coli (EPEC) and the nonpathogenic E. coli strain HB101 were provided by Dr. P. M. Sherman (University of Toronto, Toronto, ON, Canada) and were cultured in Luria Bertani broth (LB) and on LB agar plates. Transformed E. coli HB101 harboring a prokaryotic enhanced green fluorescent protein (eGFP) expression vector (Clontech, Mountain View, CA) were maintained by culture in ampicillin (100 µg/ml; Sigma Chemical Co.). The concentrations of cytokines, bacteria, and pharmacological agents used are based on related studies and are specified in the figure legends. Cytokines and drugs were added into the basal compartment of the culture well as a 45 to 60 min pretreatment (unless otherwise stated), whereas the E. coli were applied to the apical surface of the epithelial monolayer to mimic appropriate routes of in vivo exposure.

Assessment of Epithelial Barrier Function
Transepithelial Electrical Resistance. T84 cells (1 x 10⁶) were seeded onto 1-cm² semipermeable filter supports (pore size, 0.4 or 3.0 µm; Costar, Corning Inc., Cornell, NY) and cultured for 7 days until the transepithelial electrical resistance (TER) of the monolayer 1000 /cm² was measured by a voltmeter and companion electrodes (Millipore, Bedford, MA). TER of each monolayer was measured before and after treatment and is expressed as the percentage of pretreatment TER values to normalize for variation in absolute values between individual monolayers (Watson et al., 2004).

Flux of Marker Molecules. After the final TER reading, 5 µCi of the paracellular permeability probe ⁵¹Cr-EDTA (molecular mass, 360 Da; Sigma Chemical Co.) was added to the apical compartment of filter-grown T84 cell monolayers, and 4 h later, duplicate 0.5-ml samples were retrieved from the basal compartment and radioactivity determined in a gamma counter. Results are expressed as counts per minute. In additional epithelial preparations, the mucosal-to-serosal flux of horseradish peroxidase (HRP; type VI molecular mass, 44 kDa; Sigma Chemical Co.) was assessed as a marker of transcellular transport. In brief, 10 µM HRP was added to the lumenal side of the epithelial layer; 2 h later, duplicate 10-µl aliquots of the basolateral culture media were mixed with 80 µg/ml o-dianisidine (Sigma Chemical Co.) in 100 µl of reaction buffer, and HRP activity was determined in a kinetic assay by measuring absorbance at 470 nm at 30-s intervals over a 2-min period. Results are expressed as the percentage recovery of HRP (Berin et al., 1999).

Bacterial Internalization and Translocation. For internalization, 1 x 10⁶ T84 cells were cultured in 12-well plates until 80% confluent (i.e., 3–4 days), at which time IFN ± pharmacological inhibitor were added to the epithelium followed by 1 x 10⁶ cfu of E. coli HB101 (in log phase of their growth) 48 h later. Sixteen hours later, the culture medium was aspirated and replaced with fresh medium containing 250 µg/ml gentamicin (Invitrogen) for 2 h to kill extracellular bacteria. Epithelial preparations were rinsed (x4) with sterile PBS (37°C), lysed in 1 ml of cold (4°C) 0.1% Triton X-100/PBS for 15 min, and the lysates were resuspended. Serial dilutions of each lysate were streaked onto LB agar plates that were incubated under aerobic conditions at 37°C for 24 h, and bacterial colonies were subsequently counted. To complement this quantitative assessment, bacteria were visualized by immunofluorescent detection. Filter-grown T84 monolayers treated with IFN ± LY294002 for 48 h were exposed to 10⁶ cfu of E. coli HB101-eGFP. Sixteen hours later, monolayers were rinsed (x4) with sterile PBS, fixed in 10% neutral-buffered formalin for 20 min, rinsed again, and treated with rhodamine-conjugated anti-E. coli antibodies (Sigma Chemical Co.). Monolayers were rinsed in sterile PBS, excised from the culture well mounts, and mounted on poly-L-lysine-coated microscope slides in antifade mounting medium (Biomedia, Foster City, CA). Collection of both en face and z series images was performed with a LSM510 laser-scanning confocal microscope (Carl Zeiss GmbH, Jena, Germany) and a Windows 2000-based computer system and LSM510 version 2.3 software. Because the monolayers are not permeabilized, the anti-E. coli antibodies are excluded from the intracellular space; therefore, internalized bacteria appear green because of eGFP expression, and extracellular bacteria appear yellow/orange (i.e., eGFP + rhodamine).

For translocation, T84 cells were cultured on porous filter supports (pore size = 3.0 µm) until monolayers were electrically confluent (i.e., TER > 1000 /cm²), treated with IFN ± pharmacological inhibitors, and 48 h later, E. coli HB101 (10⁶ cfu) were added to the apical side of the monolayer. Sixteen hours later, 10 µl of basolateral culture medium was collected, inoculated onto LB agar plates, and cultured at 37°C for 24 h (Nazli et al., 2004). Colony growth was enumerated by a semiquantitative score (0–5) reflective of a logarithmic scale: 0, no bacterial colonies; 1, 10 colonies; 2, 10 to 100 colonies; 3, >100 colonies but countable; 4, >100 colonies, uncountable, but individual colonies can be defined; and 5, bacterial lawn (individual colonies cannot be distinguished).

Western Blotting
In these experiments, 2 x 10⁶ T84 cells/well were seeded in six-well tissue culture-treated plates or filter supports and exposed to the various experimental treatments. Whole-cell lysates were prepared by scraping cells in ice-cold radioimmunoprecipitation assay buffer containing protease (Complete protease inhibitor cocktail; Roche, Indianapolis, IN) and phosphatase inhibitors (100 mM NaF and 100 mM NaVO₃; Sigma Chemical Co.) and allowing lysis to proceed for 20 min on ice, with vigorous vortexing at 10 and 20 min. Lysates were clarified by centrifugation, and the supernatant was collected and stored at -70°C. Protein concentration was determined using the Bio-Rad/Bradford microplate assay (Bio-Rad, Hercules, CA). Protein extracts (20–40 µg) in reducing-loading buffer were boiled and electrophoresed through 4 to 10% (29:1 acrylamide/bisacrylamide) SDS gels. Separated proteins were electroblotted to Immobilon nitrocellulose membrane (Millipore) and blocked in 5% non-fat powdered milk/Tris-buffered saline/Tween 20 or 5% bovine serum albumin/Tris-buffered saline/Tween 20 for 1 h. Primary antibodies used were anti-interferon-regulated factor (IRF) 1 (1:4000) and anti-actin (1:500; Santa Cruz Biotechnology, Santa Cruz, CA), anti-pSer⁴⁷³-Akt (1:1000) and anti-Akt (1:1000; Cell Signaling Technology, Danvers, MA), anti-PKC (, , ) (1:500; Upstate Cell Signaling Solutions, Charlottesville, VA), and anti-pan phospho-PKC (1:500; Cell Signaling Technology). Blots were washed and incubated with secondary antibody-HRP conjugates for 1 h (goat anti-rabbit or rabbit anti-mouse (both at 1:4000; Santa Cruz Biotechnology) and then washed extensively, and immunoreactive proteins were visualized using enhanced chemiluminescence (Amersham Pharmacia, Piscataway, NJ) and exposing the membrane to Kodak XB-1 film (Eastman Kodak, Rochester, NY) (Watson et al., 2004).

Reverse Transcriptase-Polymerase Chain Reaction
Total RNA was extracted from semiconfluent T84 cell layers (grown in six-well culture plates) ± 6 h of exposure to IFN (20 ng/ml) and reverse transcriptase-polymerase chain reaction performed using an established protocol (Watson et al., 2004) with the following primer sequences: iNOS, forward, 5'-AGCACATTCAGATCCCCAAG-3' and reverse, 5'-TCCAGGATACCTTGGACCAG-3' (product size = 298 bp); constitutive NOS, forward, 5'-CCTGACAACCCCAAGACCTA-3' and reverse, 5'-CAGCCTCTGGACAGATGTGA-3' (product size = 495 bp); and -actin as a housekeeping gene, forward, 5'-CCACAGCAAGAGAGGTATCC-3' and reverse, 5'-CTGTGGTGGTGAAGCTGTAG-3' (product size = 437 bp). Products were electrophoresed through a 2% agarose gel, and the amplified cDNA was visualized under UV light by ethidium bromide staining.

Electrophoretic Mobility Shift Assay
Nuclear extracts and electrophoretic mobility shift assays (EM-SAs) were conducted according to a previously published protocol (Ceponis et al., 2000). In brief, nuclear extracts (5–10 µg of protein) in binding buffer were incubated for 30 min with [³²P]dCTP (NEN Life Science Products, Boston, MA)-labeled oligonucleotide probe (hSIE) containing a high-affinity STAT1 binding site (5'-GTCGACATTTCCCGTAAATC-3' and 5'-TCGACGATTTACGGGAAATG-3'). Samples were electrophoresed through a nondenaturing 6% (40:1 bis/acrylamide) polyacrylamide gel for 2.5 h at 120 V, dried under vacuum at 80°C, and visualized by autoradiography after overnight exposure (-70°C) to Kodak XAR film.

Data Analysis
Data are presented as mean ± S.E.M., where n is defined as the number of experiments or epithelial preparations examined. Data were compared by analysis of variance followed by Newman-Keuls statistical comparisons or by Students' paired or unpaired t tests where appropriate. A level of statistical significant difference was accepted at p < 0.05.

	Results

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IFN-Induced Increases in Epithelial Permeability Are PI-3K-Dependent. We and others have shown that IFN-induced decreases in TER are time-dependent and are consistently and significantly reduced at 48 h post-treatment and not earlier. For instance, in a representative experiment from the current analysis, monolayers exposed to IFN (20 ng/ml) had TER values of 116 ± 14 (4 h), 131 ± 18 (8 h), 108 ± 16 (12 h), 112 ± 13 (24 h), and 50 ± 9% (48 h) of pretreatment values (mean ± S.D.; three epithelial monolayers; numbers in parentheses indicate time post-IFN). Therefore, we concentrated our mechanistic studies on the 48-h post-IFN time point.

To assess a role for PI-3K in IFN-induced increases in epithelial permeability, two well characterized inhibitors of PI-3K activity, LY294002 (LY) and wortmannin, were used. The barrier property of epithelial cell monolayers was gauged by TER and transepithelial fluxes of ⁵¹Cr-ETDA and HRP (markers of the paracellular and transcellular permeation pathways, respectively) 48 h after exposure to IFN ± a 1-h pretreatment with LY or wortmannin. Inhibition of PI-3K activity significantly reduced or prevented the increase in epithelial permeability evoked by IFN exposure, as assessed by each marker of barrier function (Fig. 1). Corroborating these observations, use of a phosphatidylinositol analog that inhibits the binding of PH domain-containing proteins to PI-3K [e.g., phosphoinositide-dependent kinase (PDK)-1] also prevented the drop in T84 monolayer TER caused by IFN (Fig. 2). Moreover, addition of LY into the culture well 3 or 6 h after IFN still prevented the drop in TER evoked by this cytokine (Fig. 3). In contrast, the drop in TER caused by infection with EPEC was unaffected by LY cotreatment (Fig. 4), indicating stimulus specificity in the control of epithelial barrier function.

View larger version (12K): [in this window] [in a new window]   Fig. 1. PI-3K activity is necessary for IFN-induced increases in epithelial permeability. T84 epithelial monolayers were grown on filter supports and treated with IFN (20 ng/ml) ± LY (n = 6–30 monolayers per condition; A) or wortmannin (W; n = 4–6 monolayers per condition; B) and TER assessed 48 h later. Cotreatment with LY (20 µM) inhibits the increases in transepithelial flux of 51Cr-EDTA (n = 9 monolayers per condition; C) and HRP (n = 6 monolayers per condition; D) observed 48 h after IFN treatment (data are mean ± S.E.M.; *, p < 0.05 compared with control (CON); #, p < 0.05 compared with LY at 5 and 20 µM; starting TER range = 1410–3560 /cm2).

View larger version (14K): [in this window] [in a new window]   Fig. 2. A phosphatidylinositol analog (PIA) inhibits IFN-induced decreases in TER. Assessment of TER 48 h after exposure to PIA revealed no change compared with pretreatment values, whereas the drop in TER observed 48 h after treatment with IFN (20 ng/ml) was blocked by PIA [data are mean ± S.E.M.; *, p < 0.05 compared with control (CON); n = 6 monolayers per condition; starting TER range = 1120–2340 /cm2].

View larger version (13K): [in this window] [in a new window]   Fig. 3. Pharmacological blockade of PI-3K activity 6 h after IFN exposure is sufficient to prevent the reduced TER. T84 epithelial monolayers were treated with IFN (20 ng/ml) followed by LY (20 µM) simultaneously (i.e., 0 h) or 3, 6, 12, and 24 h post-IFN addition, and TER was assessed 48 h after the IFN addition [data are mean ± S.E.M.; *, p < 0.05 compared with control (CON); n = 4–8 monolayers per condition; starting TER range = 1470–2440 /cm2].

View larger version (10K): [in this window] [in a new window]   Fig. 4. Epithelial barrier dysfunction induced by EPEC is not mediated by PI-3K inhibition. EPEC (5 x 107 cfu) ± LY (20 µM) were added to the apical and basolateral side of confluent T84 epithelial monolayers, respectively, and TER was assessed 24 h later (data are mean ± S.E.M.; n = 8 monolayers per condition; *, p < 0.05 compared with control; starting TER range = 1580–2460 /cm2).

It has been suggested that the IFN effect on epithelial barrier function is due to the induction and liberation of nitric oxide (NO) (Unno et al., 1997), although others have disputed this (Satake et al., 2001). Reverse transcriptase-polymerase chain reaction analysis revealed that IFN treatment evokes small increases in constitutive NOS mRNA and significant increases in iNOS mRNA in T84 epithelial cells (Fig. 5, inset), and although some debate exists relating to NO production by these cells, others have shown that they can express iNOS mRNA and protein (Hamalainen et al., 2002; Kiang et al., 2003). Despite this, the use of L-NAME and L-NMMA, two agents that block all isoforms of NOS, and L-NIL, an inhibitor that targets iNOS, all failed to ameliorate IFN-evoked reductions in TER (Fig. 5).

View larger version (26K): [in this window] [in a new window]   Fig. 5. Inhibition of constitutive or inducible NOSs does not ameliorate IFN-induced deceases in TER. Pretreatment of T84 epithelial monolayers with inhibitors of NOS, L-NAME (1 mM), L-NMMA (100 µM), and L-NIL (10 µM) did not prevent the drop in TER evoked by IFN (20 ng/ml; 48 h) [data are mean ± S.E.M.; n = 6–12 monolayers per condition; *, p < 0.05 compared with control (CON); starting TER range = 1450–2950 /cm2]. Inset, IFN treatment (20 ng/ml, 6 h) results in a subtle increase in constitutive NOS and an obvious up-regulation of iNOS mRNA expression (three separate epithelial preparations are shown per treatment, and the bands depicted are of the predicted size based on primer sequence design).

View larger version (59K): [in this window] [in a new window]   Fig. 6. Inhibition of PI-3K does not affect IFN-induced STAT1 DNA binding activity or induced IRF-1 expression. A, nuclear protein extracts from T84 epithelial monolayers treated with IFN (20 ng/ml, 30 min) displayed obvious STAT1 DNA binding on EMSA that was not affected by pretreatment with LY (20 µM) or wortmannin (W; 0.1 µM) but was reduced by aurintricarboxylic acid (50 µM) and EGCG (100 µM) (n = 3). B, under the same experimental conditions, a small increase in STAT1 serine 727 phosphorylation was induced by IFN that was unaffected by LY pretreatment (n = 3). C, IFN-evoked increased protein expression of IRF-1 was not affected by either LY or wortmannin but was reduced by EGCG treatment as determined by immunoblotting of whole-cell protein extracts (n = 2; actin is included as a protein-loading control).

The PI-3K Target AKT Is Not Involved in IFN-Induced Increases in Epithelial Permeability.

View larger version (39K): [in this window] [in a new window]   Fig. 7. AKT is not involved in IFN-induced decreases in TER. A, use of either inhibitors of AKT activity, SH-5, or API-2 (20 µM) (n = 8–12 epithelial monolayers per condition) failed to ameliorate the drop in TER observed 48 h post-IFN treatment (20 ng/ml). B, representative immunoblot of whole-cell protein extracts (n = 4), showing that although insulin-like growth factor (IGF)-1 (10 ng/ml, 15 min) results in increased phosphorylation of AKT serine 473 (p-AKT) in T84 cells, there was no consistent induction of AKT activation by IFN even with 10-fold more cytokine (i.e., 200 ng/ml) than that required to cause increased epithelial permeability.

Inhibition of PKC Isoforms Ablates IFN-Induced, But Not IL-4-Induced, Increases in Epithelial Paracellular Permeability. PKC has been implicated as a mediator of IFN-driven events (Deb et al., 2003; Ivaska et al., 2003) and in the control of epithelial paracellular permeability (Song et al., 2001; Weiler et al., 2005). As shown in Fig. 8A, addition of the pan-PKC inhibitor BIM (5 µM) to T84 epithelial cell monolayers before IFN significantly reduced the subsequent drop in TER (200 nM BIM did not ameliorate IFN-induced reductions in T84 monolayer TER). Moreover, addition of BIM to epithelial monolayers 6 h after IFN also resulted in a significant inhibition of the IFN effect; indeed, the magnitude of the preservation of epithelial barrier function was virtually identical to that observed when the PKC inhibitor was used in a 45-min treatment protocol (Fig. 8B). However, in contrast to PI-3K inhibition, the effect of PKC inhibition resulted in only partial maintenance of epithelial paracellular permeability in the face of IFN challenge (Fig. 8A). Moreover, when LY294002 and BIM were used in combination, there was total preservation of epithelial TER following exposure to IFN (Fig. 9), and this is in agreement with the effect of PI-3K inhibition alone (Fig. 1). These data suggest that PI-3K is upstream of PKC in the mediation of IFN effects on TER (a measure of paracellular permeability), or if the pathways are in parallel, then the small effect of PKC-inhibition is masked by simultaneous blockade of PI-3K activity. In addition, we should consider the possibility that BIM may not elicit a complete pharmacological blockade of PKC (noting that TER is measured 48 h after addition of the inhibitor) or that PI-3K mobilizes other signals that add to or synergize with PKC to cause the drop in TER.

View larger version (13K): [in this window] [in a new window]   Fig. 8. Inhibition of PKC activity reduces the IFN-induced decrease in TER. A, a 45-min pretreatment with the general PKC inhibitor BIM (5 µM) significantly reduced the drop in TER caused by IFN (20 ng/ml; 48 h) [data are mean ± S.E.M.; #, p = 0.004 compared with IFN using Student's paired t test (n = 19 experiments, two to three epithelial monolayers per experiment)]. In the same experiments, PI-3K inhibition by LY (20 µM) completely prevented the effect of IFN (n = 13). B, when added 6 h after IFN, bis. was equally effective in blocking the drop in TER compared with BIM pretreatment (each symbol indicates a separate experiment and is the mean value of two to three epithelial monolayers per experiment) (solid line is the arithmetic mean; *, p < 0.05 compared with control via analysis of variance; starting TER range = 940-3280 /cm2).

View larger version (12K): [in this window] [in a new window]   Fig. 9. Bar graph showing total preservation of epithelial barrier function (assessed by TER) in IFN (20 ng/ml; 48 h) exposed T84 cell monolayers cotreated with the PI-3K inhibitor LY (20µM) ± the general PKC inhibitor, BIM (5 µM) (data are mean ± S.E.M.; n = 5 experiments with 9–16 epithelial monolayers/condition; starting TER range = 1000–2250 /cm2; * and #, p < 0.05 compared with control and IFN only).

Rottlerin (5 µM) affected neither the IFN-evoked decreases in TER nor the increased bacterial transcytosis (n = 7–16 epithelial monolayers from three experiments; data not shown). Based on the selectivity of this drug, it appears that PKC is not the PKC isoform participating in IFN-induced increases in epithelial permeability. Furthermore, BIM pretreatment did not affect the drop in TER observed 24 h after treatment with IL-4, with the higher dose of the PKC-inhibitor (i.e., 5 µM) actually enhancing the drop in TER evoked by IL-4 (Table 1).

Analysis of whole-cell protein extracts from IFN (± LY)-treated filter-grown epithelia failed to reveal any consistent increase in PKC phosphorylation at 15 to 60 min or 7 h post-treatment using an antibody that detects phosphorylated PKC, I, II, and (n = 3; data not shown).

IFN Enhances Internalization of Commensal Bacteria and Transcytosis across the Epithelium via PI-3K- and PKC-Dependent Mechanisms. The observation of IFN-induced increased transepithelial flux of HRP led us to posit that exposure to IFN could result in increased apical-to-basal transit of E. coli strain HB101, again focusing on the 48-h time point. In four separate experiments, T84 epithelial cells exposed to IFN had increased numbers of intracellular bacteria because they could be cultured after gentamicin treatment that would kill extracellular organisms (Table 2). This was confirmed by immunolocalization studies (Fig. 10A). The increased bacterial internalization translated into increased transcytosis across IFN-treated epithelia. Pretreatment with LY294002 significantly reduced bacterial internalization (Fig. 10A; Table 2), and transcytosis was inhibited by pretreating the T84 epithelial cell monolayers with either LY294002 or BIM (Fig. 10, B–D). However, and in contrast to the TER, inhibition of PI-3K and PKC activity were equally effective in reducing IFN-induced bacterial transcytosis. Indeed, in nine separate experiments using 39 epithelial monolayers, not a single grade 5 was assigned to the bacterial translocation in IFN + BIM-treated epithelia (Figs. 10 and 11), suggesting that PKC has a more prominent role in the control of transcellular permeability/bacterial transcytosis compared with the regulation of the paracellular permeation pathway (Fig. 11). Moreover, addition of LY294002 or BIM to epithelial monolayers 6 h after IFN did not prevent the IFN-evoked epithelial transcytosis of E. coli HB101 (data not shown).

View this table: [in this window] [in a new window]   TABLE 2 Inhibition of PI-3K prevents IFN-induced E. coli internalization One million T84 epithelial cells were seeded into Petri dishes, and 3 to 4 days later, they were treated with 20 ng/ml IFN ± LY (20 µM) for 48 h. The medium was replaced with fresh antibiotic-free medium containing 106 cfu of E. coli HB101. Sixteen hours later, extracellular bacteria were killed by gentamicin treatment, followed by epithelial lysis and enumeration of internalized bacteria (mean colony counts from four experiments, two to three epithelial preparations per condition per experiment).

View larger version (90K): [in this window] [in a new window]   Fig. 10. PI-3K inhibition inhibits IFN-induced increased in internalization and transcytosis of noninvasive E. coli HB101 across T84 epithelial monolayers. A, representative images taken 16 h after E. coli HB101 inoculation showing extracellular (yellow/orange) and intracellular (green) bacteria under the denoted experimental conditions [IFN, 20 ng/ml, 48 h ± the PI-3K inhibitor LY (20µM); phase contrast and fluorescence microscopy overlay; white line demarcates the edge of the epithelium]. Treatment with IFN results in greater bacterial internalization (enhanced green in the image; center). B, representative experiment showing control monolayers (n = 6; i.e., each division on the agar plate) or cells treated with IFN ± LY for 48 h, at which time E. coli (106 cfu) was added to the apical aspect of the monolayers, and basolateral medium was collected 16 h later and grown overnight on LB agar plates (numbers represent the assigned score for each bacterial translocation; , contamination). C, quantification of the translocation studies, where inhibition of PI-3K (with LY) and PKC (with 5 µM BIM) was found to significantly inhibit IFN enhancement of E. coli translocation [data are mean ± S.E.M.; n = number of epithelial monolayers; *, p < 0.05 compared with control (CON); , p < 0.05 compared with IFN only]. D, percentage of bacterial translocations scored as 5 (i.e., uncountable colonies growing as a bacterial lawn) from each of the conditions shown in C.

View larger version (16K): [in this window] [in a new window]   Fig. 11. A, quantification of E. coli HB101 translocation across T84 epithelial monolayers 48 h after exposure to IFN (20 ng/ml) ± cotreatment with the PI-3K inhibitor LY (20 µM), the PKC inhibitor, BIM (5 µM), or both drugs [data are mean ± S.E.M. of four separate (numbers inside the bars indicate the number of epithelial monolayers/condition); *, p < 0.05 compared with control; , p < 0.05 compared with IFN only]. B, percentage of bacterial translocations scored as 5 (i.e., uncountable colonies growing as a bacterial lawn) from each of the conditions shown in A.

	Discussion

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Maintenance of the enteric epithelial barrier is important in digestive health, functioning to retard the movement of antigens and bacteria from the gut lumen into the mucosa. Indeed, increased epithelial permeability is associated with infectious and idiopathic enteropathies, including Crohn's disease (Lindberg et al., 1995; Söderholm et al., 2002). Inappropriate/excessive increases in epithelial permeability would be expected to promote or prolong enteric inflammation, whereas controlled increases could be beneficial, easing the passage of phagocytes and complement into the lumen of the gut. Studies with epithelial cell lines and mice reveal that IFN, which can be released in controlled immune responses or as a component of pathophysiological reactions, decreases epithelial barrier function (Cenac et al., 2004; Clark et al., 2005). Elegant studies have documented changes in epithelial tight junction protein (e.g., claudins, occludin, zona occludens-1) expression and localization following IFN exposure (±tumor necrosis factor- cotreatment) (Prasad et al., 2005; Utech et al., 2005; Wang et al., 2005). Thus, the goal of this study was to identify intracellular signaling pathways that convert IFN-IFN receptor interaction into decreased barrier function.

IFN can activate PI3K, and we implicated this enzyme in the decreases in TER caused by conditioned medium from activated immune cells (McKay et al., 2000), which contained significant amounts of IFN, and in the drop in TER evoked by IL-4 (Ceponis et al., 2000). Pharmacological inhibition of PI-3K activity blocked IFN-induced increases in epithelial permeability. STAT1 mobilization is a major signaling event in response to IFN, and PI-3K and STAT1 can cross-talk (Choudhury, 2004). However, PI-3K inhibition failed to affect IFN-induced STAT1 activation in T84 cells as assessed by DNA binding, tyrosine phosphorylation, and transcriptional activity. These data, coupled with our previous study (Watson et al., 2004), suggest that IFN regulation of epithelial paracellular permeability is not strictly STAT1-dependent. In addition, NO (iNOS is a STAT1-regulated gene) has been implicated (Unno et al., 1997) and refuted (Satake et al., 2001) as the mediator of IFN-evoked decreases in TER across Caco-2 cell monolayers; we found that three different NOS inhibitors did not prevent the IFN effects on T84 epithelial permeability. Thus, the current study, data on the effects of IL-4 on epithelial paracellular permeability and IFN regulation of tight junction protein insertion in the cell membrane all identify PI-3K as a crucial signaling molecule mediating cytokine-evoked increases in paracellular permeability. Yet, additional pathways regulating paracellular permeation exist since EPEC-induced reductions in TER (Fig. 4) and those caused by exposure to metabolic stress and nonpathogenic E. coli were unaffected by inhibition of PI-3K activity (Nazli et al., 2004). Therefore, blockade of epithelial PI-3K signaling could be a specific means to reduce immune-mediated epithelial barrier dysfunction in vivo.

Assessing paracellular permeability revealed that i) AKT is not required for the PI-3K-dependent IFN disruption of epithelial integrity, ii) pan-inhibition of PKC significantly reduced the effects of IFN but to a lesser degree than PI-3K inhibition, iii) inhibitors of PI-3K and PKC added to epithelia 6 h after IFN still ablated the effects of IFN on TER, and iv) inhibition of PKC pathways did not affect IL-4-evoked reductions in TER.

AKT is an important mediator of many PI-3K-dependent events, but although LY294002 completely prevented IFN-induced decreases in TER, inhibitors of AKT activity failed to influence the IFN effect. Although novel, PI-3K-dependent, AKT-independent signaling is not unique; addition of IFN to erythroid progenitors resulted in a PI-3K-sensitive induction of Bcl-x expression, whereas AKT phosphorylation remained unaltered (Paiboonsukwong et al., 2003). Although we have no evidence in support of ATK involvement in the IFN disruption of epithelial integrity, inhibition of AKT activity is beneficial in preventing deceases in barrier function where apoptosis is involved (Ginzberg et al., 2004). Apoptosis does not play a prominent role in IFN evoked increases in epithelial permeability (Bruewer et al., 2003; Watson et al., 2004).

Protein kinase C can be activated in response to IFN (Deb et al., 2003; Ivaska et al., 2003), and BIM consistently reduced IFN-evoked reductions in TER. This finding is in accordance with PKC-regulation of epithelial barrier formation and maintenance, where, to date, the , , , , and isoforms of PKC have been implicated (Tomson et al., 2004; Banan et al., 2005; Weiler et al., 2005). Immunoblotting did not show increased PKC, , or phosphorylation 15 to 60 min or 7-h post-IFN treatment. The latter is consistent with the pharmacological data (i.e., rottlerin experiments), but one would have predicted PKC or PKC activation because BIM blocks these isoforms. However, PKC activation could occur at intermediate time points (2–6 h; see below); indeed, IFN can elicit multiphasic PKC activation (Mattila et al., 1993). In addition, the LY294003 + BIM treatment, like exposure to LY294002 alone, resulted in complete ablation of the ability of IFN to reduce TER. This suggests that either PI-3K is upstream of PKC or, if the pathways are distinct, that the smaller effect of PKC inhibition is overwhelmed by blocking PI-3K. Clearly, PI-3K-dependent and PKC-independent control of epithelial paracellular permeability occurs since LY294002 prevents IL-4-induced reductions in TER (Ceponis et al., 2000), whereas BIM does not (Table 1). Finally, a MAPK cascade is activated in response to IFN, but we found no involvement of either extracellular signal-regulated kinase 1/2 or p38 MAPK in IFN-evoked increases in paracellular permeability (Watson et al., 2004).

The fact that the inhibitors of PI-3K and PKC activity can be added to the epithelium up to 6 h after IFN and still block the decreased TER is intriguing. Several hypotheses explain these findings, including synthesis and release of a mediator that feeds back onto the enterocyte to activate PI-3K, biphasic PI-3K and PKC activation (Marino et al., 2003; Condliffe et al., 2005), or a delayed receptor trans-activation event, analogous to that presented for cholinergic and EGF control of epithelial Cl^- secretion (Keely et al., 1998). Thus, although the intricacies of IFN-PI-3K-PKC signaling (and parallel pathways) in the control of epithelial paracellular permeability are yet to be fully defined, there seems to be a window of opportunity in which inhibition of immune-mediated increases in epithelial permeability would be a feasible therapeutic option.

Extensive efforts have been devoted to understanding the structure and regulation of the epithelial tight junction because this is a site of vulnerability that can be exploited by pathogens (Philpott et al., 1996). However, noninvasive bacteria can cross the epithelium via a transcellular route. For instance, translocation of E. coli (strain HB101) across metabolically stressed T84 cell monolayers was dependent on a functional cytoskeleton (Nazli et al., 2005), IFN promoted the transcytosis of E. coli (strain C25) across monolayers of human Caco2 epithelia (Clark et al., 2005), and TLR4 was implicated in E. coli (strain DH5a) internalization into the IEC-6 rat epithelial cell line (Neal et al., 2006). E. coli HB101 is noninvasive and minimal translocation occurs across naive epithelial monolayers (Philpott et al., 1996). Here, initial assessment revealed greater numbers of E. coli HB101 inside IFN-treated T84 cells, and this was complemented by semiquantitative analyses showing a significant increase in bacterial transcytosis. This is a significant observation since E. coli HB101 cannot invade the enterocyte, and in the context of inflammatory bowel disease where a component of the commensal microflora (Darfeuille-Michaud et al., 2004) has been implicated in disease etiology. Inhibition of PI-3K or PKC significantly reduced IFN-driven E. coli HB101 internalization and transcytosis, underscoring the importance of maintaining the transcellular barrier function of the epithelium. These transcytosis studies revealed two additional noteworthy points. First, unlike TER, inhibition of PI-3K and PKC reduced IFN-induced bacterial transcytosis to a similar degree, implying a more prominent role for PKC in transcellular rather than paracellular permeability in this model. Second, addition of the PI-3K and PKC inhibitors 6 h after IFN did not significantly reduce the increased bacterial transcytosis. Thus, although PI-3K and PKC are important regulators of epithelial barrier function, their relative importance varies depending on whether paracellular or transcellular permeability is considered. This highlights the complexity, and by inference, the importance of appropriate control of the epithelium's barrier function.

In summary, exposure to IFN mobilizes STAT1, PI-3K, PKC, and MAPKs that culminate in the regulation of up to 500 genes and significant physiological changes. Employing human T84 epithelial cells, we show that increases in epithelial permeability evoked by IFN are PI-3K- and PKC-dependent events (Fig. 12). Moreover, and unexpectedly, inhibition of PI-3K and PKC can be delayed for up to 6 h after IFN exposure and still result in a significant amelioration of the increased paracellular, but not transcellular, permeability. Identification of specific PI-3K and PKC isoforms that regulate epithelial permeability is a major undertaking and is the next vital step as we seek to precisely delineate cytokine control of the epithelial barrier. Likewise, additional steps leading from IFN-IFN receptor interaction to increased endocytosis and reduced expression/altered localization of tight junction proteins need to be elucidated. Although many issues remain, PI-3K and PKC have been identified as crucial regulators or epithelial permeability and may represent epithelial-specific targets in the treatment of immune-mediated decreases in epithelial barrier function.

View larger version (47K): [in this window] [in a new window]   Fig. 12. Diagrammatic hypothetical schema of the intracellular signaling pathways mobilized in epithelial cells in response to IFN that affect epithelial paracellular and transcellular permeability. In response to IFN, PI-3K is activated, and in the absence of detectable Akt activation, PKC is mobilized via either PDK1 or an intermediate molecule. PI-3K, either directly and/or via PKC, and possibly other unidentified intermediates, regulates the transcription and translation of tight junction (TJ) proteins, or regulators thereof, and/or affects the trafficking and insertion of TJ proteins into the junction complex. Simultaneously PI-3K and PKC activation allow for enhanced entry of noninvasive bacteria into the enterocyte and transcytosis across the cell. STAT1 is activated in response to IFN, and although capable of leading to increased NO, NO appears not to be involved in the increased paracellular permeability in this model system (CHX, cycloheximide; IFNGR, IFN receptor; iNOS, inducible nitric oxide synthase; , phosphorylation).

	Footnotes

 
This work was supported by the Canadian Institutes of Health Research to DMM (Grant MT-13421 to D.M.M.). D.M.M. holds a Canada Research Chair (Tier 1) in Intestinal Immunophysiology in Health and Disease and is an Alberta Heritage Foundation for Medical Research scientist. J.L.W. was a recipient of a Canadian Institutes of Health Research studentship.

Article, publication date, and citation information can be found at http://jpet.aspetjournals.org.

doi:10.1124/jpet.106.113639.

ABBREVIATIONS: IFN, interferon; STAT, signal transducer and activator of transcription; PI-3K, phosphatidylinositol 3'-kinase; PKC, protein kinase C; IL, interleukin; LY294002, 2-(4-morpholinyl)-8-phenyl-1(4H)-benzopyran-4-one hydrochloride; EGCG, (-)-epigallacatechin gallate; L-NAME, N-nitro-L-arginine methyl ester; NO, nitric oxide; NOS, nitric-oxide synthase; iNOS, inducible NOS; BIM, bisindolylmalemide I; L-NMMA, N^G-monomethyl-L-arginine; L-NIL, N⁶-(1-iminoethyl)-lysine HCl; EPEC, enteropathogenic E. coli; LB, Luria Bertani broth; eGFP, enhanced green fluorescent protein; TER, transepithelial electrical resistance; HRP, horseradish peroxidase; PBS, phosphate-buffered saline; IRF, interferon-regulated factor; iNOS, inducible nitric oxide synthase; EMSA, electrophoretic mobility shift assay; LY, LY294002; PDK, phosphoinositide-dependent kinase; MAPK, mitogen-activated protein kinase; PIA, phosphatidylinositol analog; Akt, protein kinase B; SH5, Akt inhibitor II; API, Akt inhibitor V, Tricirbine; bp, base pair.

Address correspondence to: Dr. Derek M. McKay, Gastrointestinal Research Group, Department of Physiology and Biophysics, 1877 HSc, University of Calgary, 3330 Hospital Drive NW, Calgary, Alberta, Canada T2N 4N1. E-mail: dmckay{at}ucalgary.ca

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