Enterotoxigenic Escherichia coli (K88) induce proinflammatory responses in porcine intestinal epithelial cells

Developmental and Comparative Immunology 34 (2010) 1175–1182

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Enterotoxigenic Escherichia coli (K88) induce proinflammatory responses in porcine intestinal epithelial cells Bert Devriendt a,∗ , Edith Stuyven a , Frank Verdonck a,b , Bruno M. Goddeeris a,c , Eric Cox a a b c

Laboratory of Immunology, Faculty of Veterinary Medicine, Ghent University, Salisburylaan 133, B-9820 Merelbeke, Belgium Ablynx nv, Technologiepark 21, 9052 Zwijnaarde, Belgium Department Biosystems, Division Gene Technology, Faculty of Bioscience Engineering, KULeuven, Kasteelpark Arenberg 30, 3001 Heverlee, Belgium

a r t i c l e

i n f o

Article history: Received 22 February 2010 Received in revised form 11 June 2010 Accepted 12 June 2010 Available online 26 June 2010 Keywords: F4 fimbriae Flagellin Enterotoxigenic Escherichia coli Pig Intestinal epithelial cells Cytokines

a b s t r a c t Infections with F4+ enterotoxigenic Escherichia coli (ETEC) causes severe diarrhoea in piglets, resulting in morbidity and mortality. F4 fimbriae are the key virulence factors mediating the attachment of F4+ ETEC to the intestinal epithelium. Intestinal epithelial cells (IEC) are recently being recognized as important regulators of the intestinal immune system through the secretion of cytokines, however, data on how F4+ ETEC affect this cytokine secretion are scarce. By using ETEC strains expressing either polymeric, monomeric or F4 fimbriae with a reduced polymeric stability, we demonstrated that polymeric fimbriae are essential for adhesion to porcine IEC and the secretion of IL-6 and IL-8 by IEC. Remarkably, this cytokine secretion was not abrogated following stimulation with an F4-negative strain. Since this strain expresses flagellin, TLR5 mediated signalling could be involved. Indeed, porcine IEC express TLR5 and purified flagellin induced IL-6 and IL-8 secretion, indicating that, as for other pathogens, flagellin is the dominant virulence factor involved in the induction of proinflammatory responses in IEC. These results indicate a potential mucosal adjuvant capacity of ETEC-derived flagellin and may improve rational vaccine design against F4+ ETEC infections. © 2010 Elsevier Ltd. All rights reserved.

1. Introduction Enterotoxigenic Escherichia coli (ETEC) are an important cause of diarrhoea in man and animals. Mainly children in and travellers to developing countries are affected by ETEC-induced diarrhoea (Clarke, 2001; Ratchtrachenchai et al., 2004; Qadri et al., 2005). In neonatal and recently weaned piglets, ETEC-associated diarrhoea results in morbidity and mortality (Gyles, 1994) and is considered as one of the economically most important diseases in swine husbandry (Chen et al., 2004; Frydendahl, 2002; Van den Broeck et al., 1999c). ETEC express long, proteinaceous appendages or fimbriae on their surface, which mediate adhesion to the gut epithelium. Porcine ETEC strains isolated from diarrheic pigs express 5 different fimbriae of which F4 and F18 fimbriae are the most prevalent (Fairbrother et al., 2005). F4 fimbriae are composed of a major structural subunit, FaeG, and some additional minor subunits. In contrast to most fimbriae, where the adhesin is located at the tip of the fimbriae, the major subunit FaeG also functions as the adhesin (Bakker et al., 1992). Attachment of F4+ ETEC to the host epithelial cells is mediated by an interaction of F4 fimbriae with F4-specific receptors (F4R) present on the brush borders of the small intestinal entero-

∗ Corresponding author. Tel.: +32 092647339; fax: +32 092647779. E-mail address: [email protected] (B. Devriendt). 0145-305X/$ – see front matter © 2010 Elsevier Ltd. All rights reserved. doi:10.1016/j.dci.2010.06.009

cytes, enabling colonization of the small intestine (Van den Broeck et al., 1999b). Subsequently, heat-labile (LT) and heat-stable (STa/b) enterotoxins are secreted, which induce severe diarrhoea. Since F4 fimbriae are a key virulence factor involved in mediating attachment, they are an important target in vaccination studies against F4+ ETEC (Cox et al., 2002). Indeed, oral immunization of F4R+ piglets with purified F4 fimbriae induces an F4-specific intestinal immune response, which protects them against a subsequent ETEC challenge (Van den Broeck et al., 1999a; Verdonck et al., 2004a). Furthermore, the presence of the F4R is a prerequisite for the successful immunization of piglets, indicating that receptor-mediated binding is important for the induction of a protective intestinal immunity (Van den Broeck et al., 1999a). The strong immunogenicity of F4 fimbriae can be explained by their resistance to digestive enzymes, their pH stability and their polymeric nature (Snoeck et al., 2004; Verdonck et al., 2008). Indeed, oral immunization with F4 fimbriae purified from F4+ ETEC mutants, in which the polymeric stability of the fimbriae is disrupted, resulted in reduced mucosal immune responses (Joensuu et al., 2006; Verdonck et al., 2008). Intestinal epithelial cells (IEC) are pivotal for the activation of innate immunity and subsequently for the induction of adaptive immune responses (Sansonetti, 2004). IEC function as sensors detecting pathogen-associated molecular patterns (PAMPs) through pathogen-recognition receptors (PRRs), such as Toll-like receptors (TLRs). Upon recognition of these PAMPs, IEC secrete

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Table 1 Overview of the different bacterial strains. Bacterial strains GIS26 IMM01 5/95a GIS26FaeG pHMM02b HB101c a b c

Genotype of phenotype +

Reference +

+

+

+

Wild-type F4 ETEC reference strain (O149:F4ac LT STa STb ) Wild-type F4+ ETEC strain which lacks flagellin expression (O147:F4ac+ LT+ STb+ ) Wild-type F4+ ETEC strain which also lacks flagellin expression (O149:F4ac+ LT+ STb+ ) F4 deficient mutant GIS26 strain (FaeG::Cm) GIS26FaeG transformed with pHMM02 to complement F4 fimbrial synthesis E. coli K12 laboratory strain

Verdonck et al. (2004b) Verdonck et al. (2004b) Verdonck et al. (2004b) Verdonck et al. (2008) Verdonck et al. (2008)

Strain 5/95 is a Finnish field isolate and was kindly provided by Dr. J. Joensuu. Two mutations were inserted into the faeG gene, resulting in a reduced stability of the F4 fimbrial structure. Genotype: supE44, (mrcC-mrr), recA13, ara-14, proA2, lacY1, galK2, rpsL20, xyl-5, mtl-1, leuB6, thi-1.

several cytokines and chemokines, thereby alerting the underlying mucosal immune cells, such as dendritic cells, to trigger innate immune defences and promote adaptive immune responses (Kagnoff and Eckmann, 1997; Neutra and Kozlowski, 2006). However, studies on the influence of F4+ ETEC on the innate immune functions of IEC are limited. This incited us to elucidate how the polymeric nature of F4 fimbriae influences bacterial adhesion to porcine IEC and subsequently, the cytokine secretion profile of IEC in an in vitro IPEC-J2 culture system. IPEC-J2 cells provide a relevant model for intestinal epithelial cells since they form apical microvilli, express tight junction proteins, produce glycocalyx bound mucins and glycoproteins for bacterial adhesins, and are known to express cytokines and chemokines after bacterial stimulation (Burkey et al., 2006; Schierack et al., 2006; Skjolaas et al., 2007). In addition, F4+ ETEC can bind to IPEC-J2 (Koh et al., 2008; Johnson et al., 2009). Moreover, the IPEC-J2 cell line was derived from the porcine jejunum and the jejunal Peyer’s patches are the major inductive site for F4+ ETEC specific immune responses (Snoeck et al., 2006). 2. Materials and methods

other ETEC isolates (Verdonck et al., 2004b). The GIS26faeG strain was generated by inserting a chloramphenicol (Cm) resistance gene into the faeG gene (faeG::Cm), resulting in a defective F4 fimbrial biosynthesis. In the pHMM02 strain, F4 fimbrial biosynthesis was restored through the introduction of an expression vector containing the faeG gene. However, two mutations were inserted in this sequence, substituting the wild type AA by the 5/95 strain-specific AA (Verdonck et al., 2008). 2.2. Agglutination test The agglutination assay was carried out as previously described (Hu et al., 2009). Briefly, the bacterial strains were grown in Brain Heart Infusion (BHI; Oxoid, Hampshire, UK) overnight at 37 ◦ C, 200 rpm and diluted 1/2 in PBS. Next, 10 ␮l of the bacterial suspension was applied on a glass side, after which 10 ␮l of the FaeG-specific mAbs IMM01 and IMM09, generated at our laboratory, were added and mixed. IMM01 detects both FaeG monomers and polymers, whereas IMM09 only detects FaeG polymers. Visible agglutination of the bacteria after 5 min incubation was considered as positive.

2.1. Bacterial strains 2.3. Purification of ETEC virulence factors The bacterial strains used in this study are listed in Table 1. GIS26, IMM01 and 5/95 are all wild type F4+ ETEC strains. The amino acid sequences of the FaeG subunit of GIS26 and IMM01 are 100%, while the FaeG amino acid (AA) sequence differs at 7 positions between the GIS26 and the 5/95 strain. Two of these AA are specific for the 5/95 strain, while the other different AA are also found in

F4 fimbriae were purified from the E. coli strain IMM01 (F4IMM01 ), 5/95 (F45/95 ) and pHMM02 (F4pHMM02 ) as previously described (Van den Broeck et al., 1999a; Verdonck et al., 2008). Briefly, bacteria were grown in Tryptone Soya Broth (TSB; Oxoid) for 18 h at 37 ◦ C and 85 rpm. Subsequently, the F4 fimbriae were isolated from the bacteria by mechanical shearing. After precipitation through the addition of ammonium sulphate (40%, w/v), the fimbrial proteins were dialyzed, filtrated and stored at −20 ◦ C. Flagellin was isolated from strain GIS26faeG using the same protocol as to purify F4 fimbriae (Verdonck et al., 2008). The purity of the isolated fimbriae and flagellin was assessed by SDS-PAGE and Coomassie staining (Fig. 1). To confirm binding of the purified F4IMM01 fimbriae to IPEC-J2 cells, these fimbriae were conjugated with 5(6)carboxyfluorescein-N-hydroxysuccinimide ester (FluoS, 480 Da) using the fluorescein labelling kit (Roche Diagnostics, Basel, Switzerland). Confluent IPEC-J2 cells were dislodged with sterile PBS + 1 mM EDTA (PBS/EDTA) and 5.0 × 105 cells were incubated at 4 ◦ C for 30 min with 0, 5 and 25 ␮g F4IMM01 -FluoS. After removal of unbound F4IMM01 -FluoS by washing, data were acquired on a FACSCanto flow cytometer with a minimum event count of 30,000 and analysed with FACSDiva® software (Becton Dickinson, Erembodegem, Belgium). 2.4. Cell lines and culture conditions

Fig. 1. Analysis of the purity of F4 fimbriae and flagellin preparations. Heatdenatured samples (3 ␮g total protein) were loaded on a 12% SDS-PAGE in sample buffer. The migrating bands were visualized with Coomassie staining. The molecular weight marker (kDa) is shown on the left. Lanes: (1) F4IMM01 , (2) F45/95 , (3) flagellin, and (4) F4pHMM02 .

The IPEC-J2 cell line is a non-transformed intestinal epithelial cell line derived from the jejunal epithelium of a neonatal, unsuckled piglet. The cell line is maintained as a continuous culture

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(Berschneider, 1989) in Dulbecco’s Modified Eagle medium/F12 (DMEM/F12 1:1; Gibco, Merelbeke, Belgium), supplemented with 5% fetal calf serum (FCS), 2 mM l-glutamin (Gibco), 5 ␮g/ml insulin, 5 ␮g/ml transferrin, 5 ng/ml selenium (ITS, Gibco), 100 U/ml penicillin and 100 ␮g/ml streptomycin (P/S; Gibco) and 5 ng/ml epidermal growth factor (EGF; Invitrogen, Merelbeke, Belgium) at 37 ◦ C and 5% CO2 in a humidified atmosphere. These undifferentiated cells reach confluence after 3–4 days, after which the cell line is subcultured with PBS containing 0.25% trypsin (Gibco), P/S and 0.5 mM EDTA. The Caco-2 cell line (kindly provided by Prof. Dr. C. Cuvelier) was maintained in DMEM supplemented with 20% FCS, 10 mM HEPES (Gibco) and P/S at 37 ◦ C and 5% CO2 in a humidified atmosphere.

2.5. Expression of TLR5 by porcine and human intestinal epithelial cells IPEC-J2 and Caco-2 cells, grown in culture flask until confluence, were collected following dislodging of the cells with sterile PBS/EDTA. Cells were centrifugated (4 ◦ C, 400 × g, 10 min) and resuspended in radio immunoprecipitation (RIPA) buffer (1% (v/v) Triton-X-100, 1% (w/v) deoxycholic acid, 0.1% (w/v) sodium dodecyl sulphate (SDS), 50 mm Tris pH 7.4, 150 mm NaCl, 1 mM phenylmethylsulphonyl fluoride) and stored at −20 ◦ C. Cell lysates were centrifugated (10,000 × g, 4 ◦ C, 15 min) and the protein concentration of the supernatants were determined with the bicinchoninic acid reaction (BCA; Thermoscientific, Doornik, Belgium) using bovine serum albumin as a standard. Equal amounts of heatdenatured total protein (30 ␮g) in sample buffer were separated by SDS-PAGE (12%) and transferred to polyvinylidene fluoride membranes (Millipore, Brussels, Belgium) according to standard procedures. Blots were blocked overnight at 4 ◦ C in PBS with 0.5% Tween® 80 and 5% skimmed milk powder. After washing with PBS, 0.2% Tween® 20, the blots were incubated with rabbit antihuman TLR5 (1/50 dilution) (clone H-127, Santa Cruz Biotech., Santa Cruz, CA, USA) and anti-rabbit IgG conjugated to horse-radish peroxidase (HRP) (1/250 dilution) (Dako, Glostrup, Denmark). Subsequently, HRP was visualized with 3-amino-9-carbazole (AEC) staining and scanned for image processing with Adobe Photoshop.

2.6. IPEC-J2 differentiation To induce differentiation, IPEC-J2 cells were seeded at a density of 5.0 × 105 cells/well on collagen-coated 6-well transwell inserts (pore size 0.4 ␮m; 4.67 cm2 ; Corning, Amsterdam, The Netherlands). Cells were maintained in IPEC-J2 culture medium for 24 h, after which the medium was replaced by IPEC-J2 culture medium without FCS. This differentiation medium was changed every other day. To monitor differentiation, the trans-epithelial electrical resistance (TEER) was measured daily using a Millicell Electrical resistance system (Millipore). To confirm differentiation, the paracellular transport of HRP (Sigma, Bornem, Belgium) through IPEC-J2 monolayers in transwell inserts was assessed. Briefly, 75 ␮g HRP dissolved in differentiation medium was added to the apical compartment of the transwell inserts. The basolateral medium was collected at different time points, serially diluted in PBS and the amount of HRP present in the basolateral compartment was assayed by adding 2,2 -azinobis(3-ethylbenzthiazoline-6-sulphonic acid) (ABTS) as a substrate for HRP. The plates were incubated at 37 ◦ C for 30 min where after the optical density (OD) was read at 405 nm. HRP was used to create a standard curve to quantify the amount of basolateral HRP.

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2.7. Quantitative bacterial adherence assay IPEC-J2 cells were seeded at 2.0 × 105 cells/well in a 24-well culture plate (Nunc, Roskilde Denmark) and grown to 100% confluence. Prior to ETEC infection the culture medium was replaced with differentiation medium without antibiotics. Bacteria were grown overnight at 37 ◦ C in 3 ml BHI medium supplemented with the appropriate antibiotics in polystyrene tubes while shaking at 200 rpm, where after the OD660 nm was measured to determine the number of bacteria. An OD660 nm equalling 1 corresponds to 1.0 × 109 bacteria/ml. The growth rates of the bacterial strains were not significantly different. Subsequently, 1.0 × 105 bacteria in IPECJ2 differentiation medium without antibiotics were added to the IPEC-J2 monolayers in duplicate and incubated for 2 h 30. Next, the adherent bacteria were enumerated by washing them three times with PBS to remove non-adherent bacteria, followed by dislodging of the monolayers with 0.25% trypsin for 15 min at 37 ◦ C and plating serial dilutions on BHI agar plates. After overnight incubation at 37 ◦ C the number of colony forming units (CFU) were counted. 2.8. Cytokine expression Differentiated IPEC-J2 cells were incubated in triplicate either with the different bacterial strains (Table 1) or with purified bacterial virulence factors. Incubation with the bacterial strains occurred for 2 h at 37 ◦ C in a humidified atmosphere at 1.0 × 108 CFU/transwell, corresponding to a multiplicity of infection (MOI) of around 75, where after the bacteria were removed by washing three times with sterile PBS. The IPEC-J2 monolayers were further incubated for another 24 h in differentiation medium supplemented with 50 ␮g/ml gentamycin. Subsequently, both the apical and basolateral medium were collected and stored at −20 ◦ C until further processing. The IL-6, IL-8, IL-1␤ and TNF␣ concentrations were assessed in both the apical and basolateral medium with commercially available porcine-specific ELISA kits (Duoset; R&D systems, Abingdon, UK) according to the manufacturer’s guidelines. The cytokine concentrations were calculated using DeltaSOFT JV 2.1.2 software (BioMetallics, Princeton, NY, USA) with a 5-parameter curve-fitting algorithm applied for standard curve calculations. For the virulence factors, differentiated IPEC-J2 monolayers were incubated with 50 ␮g/ml purified F4IMM01 , F45/95 , F4pHMM02 , and flagellin or were left untreated. These virulence factors were diluted in IPEC-J2 differentiation medium supplemented with 100 ␮g/ml polymyxin B (Sigma) to block the activation of cells through LPS. After 24 h incubation at 37 ◦ C, the apical and basolateral IL-6 and IL-8 concentration was determined as described above. 2.9. Statistical analysis The difference in adherence of the ETEC strains to IPEC-J2 cells and cytokine secretions were analysed with SPSS 16 using one-way ANOVA and least significant difference post hoc adjustments after square root transformation of the data to homogenize variances. A p-value < 0.05 was considered statistically significant. 3. Results 3.1. F4 fimbriae mediate ETEC adherence to intestinal cells To examine if ETEC are able to adhere to IPEC-J2 cells, the intestinal cells were infected with 1.0 × 105 bacteria (Table 1) and subsequently processed to quantify the adherence of the different ETEC strains (Fig. 2). As expected both the wild type F4+ ETEC

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Fig. 2. Polymeric F4 fimbriae mediate bacterial adherence to IPEC-J2 cells. Upon reaching confluence, cells were incubated for 24 h in differentiation medium, where after 1.0 × 105 CFU of each bacterial strain, as indicated in the x-axis, were added to the cells for 2 h 30. The bacterial adherence was determined as the percentage binding and calculated as followed: (CFUbound /CFUadded ) × 100. Data are represented as the mean % binding ± SEM from 3 independent experiments (n = 3). *, p < 0.05; **, p < 0.01.

strain GIS26 and the IMM01 strain demonstrated a strong adhesion to the IPEC-J2 cells. In contrast, the absence of F4 fimbriae in the GIS26faeG strain resulted in a significantly lower adherence to the cells in comparison to GIS26 (p = 0.002) or IMM01 (p = 0.005) (Fig. 2). To elucidate the influence of the polymeric nature of the F4 fimbriae on bacterial attachment, two ETEC strains (5/95 and pHMM02) were analysed for their adherence. While both purified F45/95 and F4pHMM02 fimbriae migrate as FaeG monomers in SDSPAGE without heat treatment of the samples (Van Molle et al., 2007; Verdonck et al., 2008), only the purified F4pHMM02 fimbriae were recognized by the IMMO9 mAb in an FaeG-specific ELISA. Moreover, the length of the F4pHMM02 polymers seems to be reduced, indicative of a reduced polymeric stability of the F4pHMM02 fimbriae (Verdonck et al., 2008). Previous experiments demonstrated that purified F45/95 fimbriae consist as monomers, however, the presence of monomeric F45/95 on the surface of the 5/95 strain has never been shown (Verdonck et al., 2008). Indeed, agglutination of the ETEC strains with the IMM09 mAb (Table 2), which detects only polymeric F4 fimbriae, confirmed the presence of monomeric F4 on the surface of the 5/95 strain. Moreover, this strain showed a significantly reduced ability to adhere to the IPEC-J2 cells as compared to GIS26 (p = 0.001) and IMM01 (p = 0.002). On the other hand, the pHMM02 strain demonstrated an intermediate binding profile to the IPEC-J2 cells with a significantly reduced adherence as compared to GIS26 (p = 0.049), while in comparison to the 5/95 strain the attachment was significantly higher (p = 0.02). Altogether, these data indicate that the presence of stable polymeric F4 fimbriae on the bacterial surface is essential for F4+ ETEC attachment to intestinal epithelial cells.

Table 2 Agglutination of the different ETEC strains with two FaeG-specific mAbs, IMM01 and IMM09. The mAb IMM01 recognizes both FaeG monomers and polymers, while IMM09 only detects FaeG polymers. Bacterial strain

GIS26 IMM01 GIS26faeG pHMM02 5/95 n = 2.

mAb IMM01

IMM09

+ + − + +

+ + − + −

Fig. 3. IPEC-J2 cells differentiate following seeding on collagen-coated transwells. (A) IPEC-J2 cells (5.0 × 105 ) were seeded on collagen-coated 6-well transwell inserts. The differentiation of the monolayer was assessed by measuring the trans-epithelial electrical resistance (TEER). The data are represented as the mean TEER ± SD (n = 20) and are representative for all experiments with differentiated IPEC-J2 cells. (B) IPECJ2 cells decrease the paracellular transport of HRP upon differentiation. IPEC-J2 cells were seeded on transwell inserts and after reaching confluence, 75 ␮g HRP was added to the apical compartment and incubated for 2, 4 or 6 h, after which the amount of HRP in the basolateral compartment was measured. TEER values (x-axis) were monitored to assess differentiation. The data are represented as the mean ± SEM of 6 replicates and are representative for two different experiments.

3.2. ETEC induce IL-6 and IL-8 cytokine secretion by intestinal cells Previous studies have shown that IPEC-J2 cells are able to differentiate with a concomitant expression of tight junction proteins and high TEER values (Schierack et al., 2006; Skjolaas et al., 2007). To confirm the differentiation of IPEC-J2 cells, grown on collagencoated 6-well transwell inserts, the TEER was measured. After 5 days of culture, we observed a rise in the TEER value, which reached its maximum at days 6–7 post-seeding, indicating that the IPECJ2 cells were differentiated (Fig. 3A). Subsequently, TEER values gradually dropped back to baseline values at day 12 post-seeding, probably reflecting a decreased expression of tight junction proteins by the monolayers. Moreover, the paracellular transport of HRP (44 kDa) decreased with increasing TEER values, further indicating the formation of tight junction complexes (Fig. 3B). Based on these results, it was decided that only a differentiated IPEC-J2 monolayers with a TEER-value of at least 5000  cm2 at 7 days postseeding should be used as a model system to study the activation of the intestinal epithelium upon ETEC stimulation. Infection of intestinal epithelial cells with the F4+ ETEC reference strain GIS26 significantly induced the apical and basolateral

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Fig. 5. IPEC-J2 cells express TLR5 protein and F4 receptors. (A) Total cell lysates (30 ␮g) were separated by SDS-PAGE and analysed by immunoblotting using an anti-human TLR5 Ab. Caco-2 cell lysates were used as a positive control for TLR5 expression. (B) F4IMM01 -FluoS binds to IPEC-J2 cells in a dose-dependent manner (n = 2).

8 secretion was reduced after stimulation of IPEC-J2 cells with the ETEC strain 5/95 in comparison to GIS26. On the other hand, the IMM01 strain, which lacks flagellin expression, induced a significantly lower basolateral IL-6 secretion than the GIS26faeG strain (p = 0.037), while the latter strain tended to induce a lower basolateral IL-6 secretion compared to the GIS26 strain (p = 0.093) and a lower basolateral IL-8 secretion in comparison to the GIS26 (p = 0.145) and GIS26faeG strain (p = 0.118). These results may suggest that the induction of IL-6 and IL-8 secretion by stimulated IEC is mediated through an interaction of ETEC flagellin with IEC as well as an interaction of polymeric F4 fimbriae with F4R present on the apical surface of the IEC. We observed no TNF␣ expression after stimulation of the IPEC-J2 cells neither with the different bacterial strains nor in the control cells (data not shown). Furthermore, the IL-1␤ secretion was unaffected following infection with the different bacterial strains (data not shown). 3.3. ETEC flagellin induces IL-6 and IL-8 secretion by IPEC-J2 cells Fig. 4. Induction of IL-6 (A) and IL-8 (B) secretion by differentiated IPEC-J2 cells after stimulation with the different ETEC strains as indicated in the x-axis. The data are represented as the mean cytokine concentration ± SEM of 5–7 independent experiments (n = 5–7). Letters indicate a statistical significance (p < 0.05) between treatment groups: (a) in comparison to control, (b) to HB101, (c) to GIS26faeG. TW, transwell.

secretion of both IL-6 (Fig. 4A) and IL-8 (Fig. 4B) as compared to non-infected cells and cells infected with the non-pathogenic E. coli strain HB101. The latter failed to induce cytokine secretion above control levels (Fig. 4A and B). Intriguingly, the absence of F4 fimbriae on the GIS26faeG strain did not abrogate the ability of this strain to induce significantly higher IL-6 and IL-8 secretion as compared to control cells (Fig. 4A and B), although a reduced binding to the IPEC-J2 cells was observed in comparison to GIS26 (Fig. 2). Complementation of F4 fimbrial synthesis in the pHMM02 strain restored apical and basolateral IL-6 cytokine secretion by IPEC-J2 cells. Although not significantly, the secretion of apical and basolateral IL-8 was reduced as compared to GIS26 and GIS26faeG (Fig. 4). In addition, both the apical and basolateral IL-6 and IL-

The induction of IL-6 and IL-8 secretion by the GIS26faeG strain was surprising, especially as a weaker binding to the intestinal cells was observed. The data above indicated that flagellin might be involved in this process. Since flagellin is recognized by Tolllike receptor (TLR) 5 (Hayashi et al., 2001) and the expression of this TLR by IPEC-J2 cells has not yet been demonstrated, we analysed TLR5 expression by IPEC-J2 cells. As shown in Fig. 5A, the TLR5 mAb detected a protein band migrating at the predicted molecular weight of porcine TLR5 (98 kDa), suggesting that IPECJ2 cells express TLR5. In line with previous reports (Johnson et al., 2009), purified F4IMM01 fimbriae were able to bind to IPEC-J2 in a dose-dependent manner (Fig. 5B), confirming the expression of F4-specific receptors by IPEC-J2 cells. To further analyse the importance of ETEC fimbriae and flagellin in the induction of IL-6 and IL-8 secretion, differentiated IPEC-J2 cells were stimulated with purified F4 fimbriae and flagellin (Fig. 6). Both the apical IL-6 (Fig. 6A) and basolateral IL-8 (Fig. 6B) expression was significantly upregulated after apical stimulation of IPEC-J2 cells with flagellin in comparison to non-stimulated cells, F4IMM01 and F4pHMM02 stim-

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Fig. 6. ETEC-derived flagellin induces apical IL-6 (A) and basolateral IL-8 (B) secretion by differentiated IPEC-J2 cells. Monolayers were stimulated with purified virulence factors (75 ␮g) for 24 h in differentiation medium supplemented with 100 ␮g/ml polymyxin B. The data represent the mean cytokine concentration ± SEM of 3 independent experiments (n = 3). The asterix indicates a statistical significance (p < 0.05) between flagellin stimulated and non-stimulated, F4IMM01 and F4pHMM02 stimulated cells. TW, transwell.

ulation. Unexpectedly, we observed no enhanced IL-6 and IL-8 secretion following stimulation of IPEC-J2 cells with F4 fimbriae, either as polymeric or monomeric proteins. Both IL-1␤ and TNF␣ were not analysed given the fact that stimulation with ETEC failed to affect the secretion of these cytokines. These results indicate that ETEC-derived flagellin enhances the cytokine secretion by intestinal epithelial cells, whereas F4 fimbriae seem unable to induce cytokine secretion. 4. Discussion In the present study, we confirmed previous in vitro and ex vivo findings that F4 fimbriae mediate adhesion of F4+ ETEC to intestinal

epithelial cells (Koh et al., 2008; Pavlova et al., 2008; Van den Broeck et al., 1999a). Indeed, we observed a significantly reduced adhesion of the ETEC mutant strain GIS26faeG, in which the assembly of the F4 fimbriae is abrogated through deletion of the faeG gene, as compared to the wild type strain GIS26. However, a low adhesion of GIS26faeG to IEC was persistently observed. This could be explained by binding of the bacteria to IEC via type I pili (Van den Broeck et al., 1999a). Alternatively, certain carbohydrate moieties on the apical surface of IEC are recognized by most E. coli, independent from the expression of known adhesion factors (Jansson et al., 2009). Intriguingly, ETEC strains in which the polymeric nature of the F4 fimbriae is affected (5/95 and pHMM02), also demonstrated a significantly reduced adhesion. These data suggest that polymeric F4 fimbriae are necessary to mediate F4+ ETEC attachment to the intestinal epithelium. In recent years, the role of IEC in the activation of innate immune responses and subsequently the induction of adequate adaptive immunity to intestinal pathogens has become increasingly evident (Neutra and Kozlowski, 2006). IEC can sense pathogens through PRRs, such as TLRs. Signalling through these receptors activates a transcriptional profile, resulting in the expression of several cytokines and chemokines, thereby attracting immune cells and alerting the intestinal immune system to an ongoing infection (Kagnoff and Eckmann, 1997). Porcine IEC have been shown to express several TLRs and to enhance cytokine mRNA expression upon pathogen or PAMP stimulation (Arce et al., 2008; Moue et al., 2008; Mariani et al., 2009; Meurens et al., 2009). However, few studies have examined the cytokine secretion response of IEC to F4+ ETEC infection. In one study, Pavlova et al. (2008) showed that F4+ ETEC failed to enhance IL-8 and TNF␣ mRNA expression by the IPI-2I cell line. This cell line is derived from the porcine ileum, a site of minor importance for F4+ ETEC pathogenesis. On the contrary, we demonstrated for the first time that infection of differentiated monolayers of IPEC-J2, which are derived from the porcine jejunum, with the wild type F4+ ETEC strain significantly enhanced apical and basolateral IL-6 and IL-8 cytokine secretion. On the contrary, the non-pathogenic E. coli strain HB101 failed to induce cytokine secretion, suggesting that the observed cytokine secretion is not merely a general inflammatory response to any bacteria, but is rather specific for ETEC infection. IL-6 is known to stimulate neutrophil degranulation (Sitaraman et al., 2001), B cell class switching (Sato et al., 2003) and to modulate the maturation of dendritic cells (DC) (Horn et al., 2000), while IL-8 serves as a chemoattractant for neutrophils (Gesser et al., 1996). The enhanced secretion of IL-8 can explain the strong infiltration of neutrophils in the lamina propria upon F4+ ETEC infection (Faubert and Drolet, 1992). Since polymeric F4 fimbriae are essential for bacterial adhesion, one could envision their importance in IEC activation. Indeed, the IL-6 and IL-8 secretion by IPEC-J2 seems to be reduced upon stimulation with F4+ ETEC strains expressing monomeric fimbriae or fimbriae with a reduced polymeric stability. Since destabilization of the F4 fimbrial polymers previously resulted in reduced F4-specific immune responses following oral immunization (Verdonck et al., 2008), this suggests that F4 fimbriae have to be expressed as stable polymers on the bacterial surface in order to adequately induce IEC cytokine secretion. Consistent with earlier findings, we also observed a polarized apical IL-6 and basolateral IL-8 secretion by IEC upon pathogen stimulation (Sitaraman et al., 2001; Skjolaas et al., 2007). Apical IL-6 secretion into the lumen could serve several functions. Epithelial cells are known to express the IL-6 receptor (Keller et al., 1996), which would allow infected cells to alert adjacent cells of infection. Furthermore, during active inflammation in the human intestine, neutrophils can transmigrate to the intestinal lumen. Following IL-8 induced recruitment, apical secreted IL-6 could therefore induce neutrophil degranulation, thereby enhancing the microbicidal activity of neutrophils (Sitaraman et al., 2001).

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In addition, DC extend dendrites through the epithelial barrier to sample luminal contents and luminal IL-6 and IL-8 could activate these DC (Rescigno et al., 2001). In contrast to IL-6 and IL-8, IL-1␤ expression was unaffected by ETEC infection, while TNF␣ expression was not observed. However, LPS-activated IPEC-J2 upregulate IL-1␤ and TNF␣ mRNA expression (Arce et al., 2008). These differences could reflect discrepancies in culture conditions or translational regulation of the corresponding mRNA’s. The maximum IL-1␤ or TNF␣ response could have been missed at the time point studied, since IL-1␤ expression reached its maximum at 3.5 h following bacterial stimulation of human IEC and diminished thereafter (Bandyopadhaya et al., 2008). Alternatively, a recent study indicates that IEC require a crosstalk with APC in order to express their full cytokine potential upon bacterial stimulation (Zoumpopoulou et al., 2009). If porcine IEC respond differently to F4+ ETEC when cocultured with APC awaits further study. Surprisingly, we observed no difference between the GIS26 and GIS26faeG strains in their ability to promote apical and basolateral IL-6 and IL-8 secretion by IPEC-J2. On the contrary, the IMM01 strain, which lacks flagellin, failed to induce a similar cytokine secretion profile. Flagellin is the major structural protein of the flagellum and consists of a conserved domain that is widespread among bacterial species (Ramos et al., 2004). This PAMP is recognized by TLR5 (Hayashi et al., 2001), which is expressed on the apical and basolateral surfaces of human and murine enterocytes (Cario and Podolsky, 2000; Bambou et al., 2004). Thus, the observed IL-6 and IL-8 secretion by IEC could be explained by a TLR5 mediated signalling cascade upon flagellin detection. The fact that IPEC-J2 cells express TLR5 supports this assumption. Moreover, the induction of basolateral IL-8 secretion by flagellin stimulated porcine IEC is consistent with earlier reports in human and murine IEC (Cario and Podolsky, 2000; Sierro et al., 2001; Bambou et al., 2004). These data suggest that like for other flagellated pathogens, such as S. typhimurium, EHEC, EPEC, EAEC and V. cholerae, flagellin is the dominant IL-8 inducing ETEC virulence factor (Ramos et al., 2004). In contrast to flagellin, purified F4 fimbriae, either as polymers or monomers, were unable to upregulate cytokine expression by IEC. Nevertheless, F4 fimbriae are transcytosed across the porcine epithelium in vivo (Snoeck et al., 2008) and induce the maturation of intestinal DC (Devriendt et al., 2009). It is tempting to speculate that the putative F4 receptor involved in transcytosis does not induce cytokine secretion by IEC, but that clustering of this receptor is required for efficient transcytosis. In that way, polymeric F4 fimbriae would be transported more efficiently across the epithelial barrier, resulting in a higher local concentration of F4 fimbriae available to antigen-presenting cells. This could explain the induction of weaker mucosal immune responses following oral immunization with purified F45/95 and F4pHMM02 fimbriae (Joensuu et al., 2006; Verdonck et al., 2008). In conclusion, the results in this study point out that F4 fimbriae need to be expressed as stable multimeric proteins on the bacterial surface for efficient attachment to the epithelium and that the interaction between F4-specific receptors on the apical surface of the epithelial cells and polymeric F4 fimbriae are required to enhance the secretion of proinflammatory cytokines by the IEC. Furthermore, ETEC-derived flagellin could act as a potent mucosal adjuvant.

Acknowledgements We gratefully acknowledge S. Brabant, D. Slos and G. De Smet for their technical assistance. B. Devriendt was supported by a Ph.D. grant of the Institute for the Promotion of Innovation through Science and Technology (IWT-Vlaanderen). We thank Dr. J. Joensuu for

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