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Infection and Immunity, September 2005, p. 5514-5523, Vol. 73, No. 9
0019-9567/05/$08.00+0     doi:10.1128/IAI.73.9.5514-5523.2005
Copyright © 2005, American Society for Microbiology. All Rights Reserved.

Host Immune Status Influences the Development of Attaching and Effacing Lesions in Weaned Pigs

Francis Girard,1 Isabelle P. Oswald,2 Ionélia Taranu,2 Pierre Hélie,3 Greg D. Appleyard,4 Josée Harel,1 and John M. Fairbrother1*

Groupe de recherche sur les maladies infectieuses du porc (GREMIP),1 Département de pathologie et microbiologie, Faculté de médecine vétérinaire, Université de Montréal, Québec, Canada,3 Laboratoire de Pharmacologie-Toxicologie, INRA, Toulouse, France,2 Western College of Veterinary Medicine, University of Saskatchewan, Saskatoon, Saskatchewan, Canada4

Received 7 December 2004/ Returned for modification 4 April 2005/ Accepted 5 May 2005


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ABSTRACT
 
Attaching and effacing Escherichia coli (AEEC) has been associated with naturally occurring attaching and effacing (A/E) lesions in weaned pigs, and although A/E lesions have been experimentally reproduced in newborn piglets, such lesions have been much more difficult to induce in older conventional pigs. Hence, the aim of this study was to examine the effect of oral administration of dexamethasone on the development of A/E lesions in weaned pigs challenged with a porcine enteropathogenic E. coli (PEPEC) strain and to investigate the involvement of local intestinal cytokine response. Dexamethasone, given orally at a dosage of 3 mg kg of body weight–1, significantly enhanced both the colonization of the challenge strain and the prevalence of foci of intimately adherent bacteria, resulting in extensive A/E lesions in the ileum, cecum, and colon of challenged pigs. We also confirmed the expression of both intimin and Tir by PEPEC strain ECL1001 in A/E lesions in vivo, which is, to our knowledge, the first report of the involvement of the latter proteins in any AEEC infections in vivo. Moreover, semiquantitative reverse transcription-PCR demonstrated that interleukin 1ß (IL-1ß), IL-6, IL-8, and, to a lesser extent, IL-12p40 are significantly upregulated in the ileum following challenge with strain ECL1001, whereas dexamethasone blocks such upregulation. Taken together, our results strongly suggested that host immune status influences the development of A/E lesions in weaned pigs, and it appears that IL-1ß, IL-6, IL-8, and, to a lesser extent, IL-12p40 are expressed during infection of weaned pigs by PEPEC and may contribute to the natural resistance of the host against PEPEC infection.


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INTRODUCTION
 
Enteropathogenic Escherichia coli (EPEC) and Shiga toxin-producing E. coli (STEC) are an important cause of enteric disease in both humans (39) and other animal species, including rabbits (6), calves (27), pigs (24), and dogs (4). EPEC and some STEC are attaching and effacing E. coli (AEEC), since they cause typical attaching and effacing (A/E) intestinal lesions. A/E lesions are characterized by intimate bacterial adherence to enterocytes, effacement of surface-absorptive microvilli, F-actin rearrangement, and the outgrowth of a cuplike pedestal of polymerized F-actin and other cytoskeletal elements beneath the adherence site. Most A/E phenotype elements are encoded on a 35.6-kb (EPEC) to 43-kb (STEC of O157:H7 serotype, STECO157:H7) pathogenicity island called locus of enterocyte effacement (LEE) (35, 36). The LEE contains genes encoding an outer membrane adhesin termed intimin (eae gene), a type III secretion system machinery (Esc and Sep proteins), chaperones (Ces proteins), and translocator (EspA, EspB, and EspD) and effector (EspF, EspG, and Map) proteins, as well as the translocated intimin receptor (Tir).

AEEC has been associated with naturally occurring cases of A/E lesions in weaned pigs (27). However, although A/E lesions have been reproduced in newborn piglets using porcine EPEC (PEPEC) (24, 73) and STECO157:H7 (11, 13, 66), we have been unable to induce such lesions in older conventional pigs. Many factors may contribute to the lack of development of A/E lesions in these pigs, including a more substantial and diversified intestinal normal flora and a more mature immune system (38). Indeed, diseases associated with AEEC, such as hemolytic uremic syndrome (31) and hemorrhagic colitis (64), are most often observed in younger or older individuals with an immature or compromised immune status. Also, it appears that the gut-associated lymphoid tissue of the host immune system is an important determinant of both colonization and A/E lesion development by the related murine A/E pathogen Citrobacter rodentium in vivo (68).

Clinical and experimental observations have shown that the Porcine reproductive and respiratory syndrome virus (PRRSV), a small enveloped RNA virus from the family Arteriviridae that replicates primarily in macrophages (18), causes immunosuppression of infected pigs, resulting in increased susceptibility to several secondary bacterial diseases, such as Streptococcus suis (20) and Salmonella choleraesuis infections (71). Possible mechanisms for this immunosuppression in PRRSV-infected animals could be an inability either to produce sufficient numbers of prethymocytes or for the prethymocytes to properly target themselves to the thymus (18). It has been recently proposed that the open reading frame 5 of PRRSV would induce apoptosis of thymocytes and hence their depletion (63). Some preliminary experiments carried out in our laboratory have strongly suggested a relationship between the presence of naturally occurring PRRSV infection and an increased prevalence of A/E lesions in weaned pigs challenged with PEPEC strains (F. Girard et al., unpublished data).

Dexamethasone (DEX) is a long-acting synthetic glucocorticoid that exerts similar effects on the immune system to those of open reading frame 5 of PRRSV, primarily by suppressing innate immunity and inhibiting the production of various proinflammatory mediators, such as cytokines and chemokines. These effects are due to suppression of Th-cell responses (65) and to leukocyte death through apoptosis (8). DEX is also an anti-inflammatory agent. It has been shown to reduce leukocyte accumulation in many models (2); to inhibit release of lactoferrin, a known stimulus for polymorphonuclear leukocyte (PMN) degranulation (54); and to reduce tracheal release of interleukin 8 (IL-8) in children with respiratory syncytial virus infection (70). DEX has been used in many animal infection models to promote bacterial colonization, including a calf model for STECO157:H7 (62) and a PRRSV-S. choleraesuis dual infection model (71).

Hence, the aim of this study was to examine the effect of oral administration of dexamethasone on the development of A/E lesions in weaned pigs challenged with a PEPEC strain and to investigate the involvement of local intestinal cytokine response in the development of these lesions.


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MATERIALS AND METHODS
 
Bacterial strains. The PEPEC strain ECL1001 (formerly 86-1390) (O45:H–) was isolated at the Escherichia coli Laboratory of the Faculté de médecine vétérinaire of the Université de Montréal (Saint-Hyacinthe, Québec, Canada) from a 4-week-old pig with postweaning diarrhea (24). Strain ECL1001 possesses a ß-intimin subtype and induces mostly cecal and colonic A/E lesions in both gnotobiotic (24) and conventional neonatal (72, 73) piglets. The strain ECL1001 Nalr is a variant of strain ECL1001 that was obtained by its serial passage following growth in Luria-Bertani (LB) broth containing concentrations of nalidixic acid from 0 to 50 µg ml–1 at 37°C for 24 h. Strain ECL1001 Nalr possesses all tested LEE-associated genes.

Experimental challenge of pigs. A total of 45 weaned pigs from a conventional, certified PRRSV-negative herd were used in four separate experiments. Pigs were randomly assigned to the appropriate groups in each experiment. Pigs were weaned at 17 days of age (day 0 [D0]) onto a solid granular diet containing 19% soybean protein and no antibiotics, a diet associated with high diarrhea scores in weaned pigs challenged with ETEC strains (J. M. Fairbrother et al., unpublished data). Pigs were fed ad libitum with this diet and water for 5 days prior to bacterial infection (D0 to D5). Pigs received one of the following treatments: (i) dexamethasone (Pharmascience, Inc., Montreal, Canada) administered orally at a daily dose of 3 mg kg of body weight–1 (DEX+ groups 1, n = 20, and 3, n = 5) starting 3 days before the first bacterial challenge (D3) and continuing until necropsy (D10) or (ii) no treatment with dexamethasone (DEX– groups 2, n = 15, and 4, n = 5). On D6, D7, D8, and D9, pigs received 10 ml of 1.2% CaCO3 through an intraoesophagic tube to neutralize gastric acid. Then, pigs in groups 1 and 2 were challenged with 1 ml of 1.0 x 1010 CFU of PEPEC strain ECL1001 in 9 ml of Trypticase soy broth (TSB; Difco Laboratories, Detroit, MI) (PEPEC+) daily from D6 to D9, whereas pigs in groups 3 and 4 received 10 ml of sterile TSB broth daily (PEPEC–). None of the piglets demonstrated Nalr E. coli colonies on fecal cultures prior to bacterial challenge. Pigs were monitored daily, and general appearance, attitude, dehydration, food and water intakes, as well as diarrhea, were evaluated in accordance with the Guidelines of the Canadian Council for Animal Care.

Necropsy procedure. On D10, pigs were sedated by intramuscular injection of a mixture of 10 mg kg–1 of ketamine hydrochloride (Biomeda-MTC, Ontario, Canada) and 20 mg kg–1 of xylazine (Bayer, Ontario, Canada) before being euthanized by an intracardiac injection of sodium pentobarbital (540 mg ml–1; Pharmacie, Faculté de médecine vétérinaire, Quebec, Canada). Necropsies were performed, and a portion of the last 5 cm of the distal ileum and the antral part of the cecum and mid-spiral colon were collected and immediately fixed in 10% neutral-buffered formalin and 2.5% glutaraldehyde for examination by light microscopy and transmission electronic microscopy (TEM), respectively. Additional portions of each tissue were placed on ice for bacteriological examination, and an additional portion of ileum (containing or lacking Peyer's patches, corresponding to the antimesenteric and mesenteric surfaces of the intestine, respectively) was flash frozen in liquid nitrogen in 1 ml of TRIzol (Gibco BRL, Burlington, Ontario, Canada) for RNA extraction and analysis of cytokine gene expression. Peripheral blood was obtained from each pig by heart puncture in a tube without anticoagulant following sedation and immediately prior to euthanasia, for determination of serum cortisol. Blood samples were allowed to stand at 4°C overnight before being centrifuged at 400 x g for 10 min (59). All serum samples were immediately collected and frozen at –70°C until used.

Bacteriological counts. Tissues were evaluated quantitatively for the presence of Nalr E. coli. Samples were weighed, suspended in 1 ml of phosphate-buffered saline (PBS), homogenized at 5,000 rpm by using a CAT X-120 homogenizer (PolyScience, Niles, Ill.), and 10-fold serially diluted in sterile PBS. Appropriate dilutions were inoculated onto MacConkey agar medium containing 50 µg ml–1 nalidixic acid, using a Spiral Plater apparatus (Spiral Systems Inc, Cincinnati, Ohio). Lactose-positive colonies were counted, and the concentration of Nalr CFU per gram of tissue was calculated.

Determination of blood cortisol levels. Blood cortisol levels were measured using a solid-phase, competitive chemiluminescent enzyme immunoassay Immulite-Cortisol automated test (Diagnostic Products Corporation, California), as previously described (49). Results (in nanomoles per liter) are presented as the median for each group of pigs.

Production of antisera. Antiserum against formalinized whole bacteria of PEPEC strain ECL1001 (O45:H–) was raised in rabbits by standard techniques (17). For the production of anti-intimin (rabbit and chicken) and anti-Tir (chicken) antisera, the eae (encoding intimin) and tir genes from strain ECL1001 were amplified by PCR with the primer pairs listed in Table 1, as described previously (3). Purified His-intimin and His-Tir fusion proteins were used to immunize either rabbits (His-intimin) or laying hens (His-intimin and His-Tir). Intimin- and Tir-specific immunoglobulin Y (IgY) was then extracted from egg yolks as previously described (3), and the water-soluble fraction was lyophilized. Serum containing intimin-specific antibodies was recovered from rabbits following exsanguination and frozen at –20°C.


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  		TABLE 1. His-tagged fusion proteins used in this study

Detection of Tir secretion and intimin expression by immunoblotting. To promote Tir secretion, PEPEC strain ECL1001 was grown overnight in minimal essential medium (MEM; Gibco BRL, Ontario, Canada), transferred to 100 ml of fresh MEM, and incubated at 37°C with agitation to an optical density (OD) of 1.0 at 600 nm, as previously described (9, 28). Bacteria were pelleted by centrifugation, and phenylmethylsulfonyl fluoride (50 µg ml–1; Sigma Chemical Co., St. Louis, Mo.), aprotinin (0.5 µg ml–1; Roche Diagnostics GmbH, Manheim, Germany), and EDTA (0.5 µM; Fisher Scientific, New Jersey) were added to the supernatant. Proteins were precipitated by the addition of 10% trichloroacetic acid (Fisher Scientific, New Jersey) overnight at 4°C. After centrifugation at 4,000 x g for 1 h, protein pellets were washed with cold 95% ethanol and centrifuged again at 4,000 x g for 1 h. Pellets were resuspended into 1.0 ml of 1x Laemmli buffer and boiled for 5 min. Samples were further analyzed by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (10% acrylamide).

To promote intimin expression in whole-cell extracts of PEPEC strain ECL1001, stationary TSB cultures were diluted 1:100 in fresh DMEM and incubated for an additional three hours at 37°C with agitation. An equivalent of an OD600 of 0.1 was loaded onto 8% sodium dodecyl sulfate-polyacrylamide gel electrophoresis gels, as previously described (30).

In both cases, electrophoresed polypeptides were transferred onto a pure nitrocellulose membrane and immunoblotted with rabbit polyclonal anti-intimin (1:500), chicken polyclonal anti-intimin (1:500), or anti-Tir antiserum (1:500), followed by goat anti-rabbit-horseradish peroxidase conjugate or rabbit anti-chicken IgY-horseradish peroxidase conjugate (Bio-Rad Laboratories, Hercules, CA), respectively. The reaction was visualized using 4-chloro-1-naphthol (Sigma Chemical Co., St. Louis, Mo.).

Slide agglutination. Three colonies from MacConkey agar plates of each intestinal segment from each pig were randomly tested by slide agglutination using rabbit anti-O45K"E65" to confirm the presence of the challenge strain ECL1001.

Histopathology. Formalin-fixed tissue samples were embedded in paraffin, sectioned at 5 µm, and stained with hematoxylin, phloxine, and safranine (HPS) according to standard techniques. Two sections from each tissue sample were examined by light microscopy, and the mucosal epithelium located between adjacent crypts (designated the intercrypt mucosal epithelium [ICME]) was examined for the presence of intimately adherent bacteria. The mean percentage of ICME demonstrating intimately adherent bacteria per section was calculated. At least 40 ICME per section were examined.

Indirect immunofluorescence assay. An indirect immunofluorescence assay (IFA) was used for detection of O45-positive bacteria, corresponding to the O serogroup of the challenge strain ECL1001, and for detection of expression of intimin and Tir proteins in intestinal sections of challenged pigs. Formalin-fixed intestinal sections were deparaffinized, washed in PBS, permeabilized with 0.1% saponin (Sigma Chemical Co., St. Louis, Mo.), and treated with a blocking solution containing 10% normal donkey serum (Jackson ImmunoResearch Laboratories, Inc.) in PBS containing 1% bovine serum albumin and 1% Tween 20. After several washes in PBS-Tween 20, sections were incubated with an appropriate dilution in PBS-bovine serum albumin-Tween 20 of rabbit (O45, intimin), or chicken (Tir) primary antibodies, followed by an additional incubation step with donkey anti-rabbit Rhodamine Red-X-conjugated or donkey anti-chicken fluorescein isothiocyanate-conjugated secondary antibody, respectively (Jackson ImmunoResearch Laboratories, Inc.). DNA of epithelial cells and bacteria was counterstained with 5 µg ml–1 of 4',6-diamidino-2-phenylindole (DAPI), dilactate (Sigma-Aldrich Co., St. Louis, Mo.). Sections were mounted and examined with a Leica DMR microscope (Leica Microsystems, Wetzlar, Germany) equipped with epifluorescence and UV excitation modules (Chroma Technology Corp., Rockingham, VT).

Transmission electron microscopy. Portions of ileum, cecum, and colon were fixed for 2 h at room temperature in 2.5% glutaraldehyde and then rinsed in cacodylate buffer (0.1 M cacodylate, pH 7.3) for 1.5 h with regular changes. Portions of tissues for which typical intimately adherent bacteria were observed on light microscopy (two to three pigs per group) were postfixed for 1 h at room temperature in 2% osmium tetroxide (OsO4), rinsed in water for 1.5 h with regular changes, and dehydrated in a graded ethanol series. Tissues were then embedded in Spurr resin (Marivac, St. Laurent, Quebec, Canada). Thin sections were mounted on copper grids, stained with uranyl acetate and lead citrate, and examined for A/E lesions with a Philips 420 transmission electron microscope at 80 kV (Philips Electronics, Eindhover, The Netherlands).

RT-PCR detection of cytokine mRNA and densitometric quantification of PCR products. All ileum samples from two randomly selected experiments, conserved in TRIzol at –80°C, were homogenized using a CAT X-120 homogenizer (PolyScience, Niles, Ill.). Total RNA was extracted as recommended by the manufacturer. The RNA was resuspended in 50 to 500 µl of ultrapure water containing 0.02% (wt/vol) diethyl pyrocarbonate (Sigma, St. Quentin Fallavier, France) and 1 mM EDTA. Total RNA was quantified by using a spectrophotometer at an optical density of 260 nm (OD260), and the purity was assessed by determining the OD260/OD280 ratio. All of the samples had an OD260/OD280 ratio above 1.8. A reverse transcription-PCR (RT-PCR) procedure was performed as previously described (14). Briefly, 1 mg of RNA was reverse transcribed (Superscript II RNase H 2; Life Technologies, Eragny, France) and then amplified (Taq DNA polymerase; Promega, Charbonnières, France). Oligonucleotide primers for IL-1ß, IL-6, IL-8, and IL-12p40, tumor-necrosis factor {alpha} (TNF-{alpha}), inducible nitric oxide synthase (iNOS), and cyclophilin and the number of PCR cycles selected for each PCR amplification of porcine cytokines and cyclophilin cDNAs were described previously (14, 19, 43, 45). Amplified DNA was analyzed by electrophoresis and quantified densitometrically using the Quantity One program (Bio-Rad, Hercules, Calif.). To compare the relative cytokine mRNA expression levels among samples, the values were presented as the ratio of the band intensity of the cytokine-specific RT-PCR product over that of the corresponding constitutively expressed housekeeping gene cyclophilin.

Image capture. Images of tissue sections from histopathological and IFA examination were captured with a CCD CoolSNAP camera (RS Photometrics, California), and were computer processed with Adobe Photoshop 5.0 and Adobe Illustrator 8.0 software (Adobe Systems Incorporated, California).

Statistical analysis. Results are presented as scatter plots with the median. A Wilcoxon two-sample test (cortisol and cytokines), a mixed linear model with repeated measures (colonization), or a logistic regression model with repeated measures (prevalence) was performed with commercially available software (SAS 8.1; SAS Institute, Cary, North Carolina), and Kruskal-Wallis or Tukey posthoc comparisons were done to assess differences between the groups, respectively. A P value of <0.05 was taken to be significant.


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RESULTS
 
Confirmation by immunoblotting of the ability of the anti-intimin and anti-Tir sera to detect their homologous proteins. A 97-kDa band, corresponding to intimin, was recognized in the whole-cell extract of PEPEC strain ECL1001 on immunoblotting. An 80-kDa band, corresponding to the secreted protein Tir, and some breakdown products were revealed in the culture supernatant of the PEPEC strain ECL1001 on immunoblotting (Fig. 1).



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  		FIG. 1. Western blot analysis of whole-cell extract and secreted proteins from culture supernatants of PEPEC strain ECL1001 with either rabbit or chicken polyclonal anti-intimin antisera and chicken polyclonal anti-Tir antiserum, respectively. The whole-cell extract reacted with both intimin antisera to reveal a 97-kDa band and some breakdown products, whereas the concentrated supernatant reacted with the Tir antisera to reveal an 80-kDa band, as well as some breakdown products. Molecular mass markers are in kilodaltons.

Treatment with dexamethasone results in more extensive A/E lesions in weaned pigs challenged with the PEPEC strain ECL1001. To evaluate the role of the host immune status on the development of A/E lesions, the immunosuppressant agent dexamethasone was administered orally to weaned pigs, which were then experimentally challenged with PEPEC strain ECL1001. The level of colonization by O45-positive Nalr E. coli was significantly greater (P = 0.008) in DEX+/PEPEC+ pigs than in DEX–/PEPEC+ pigs (Fig. 2A). No O45+ bacteria were recovered from the DEX–/PEPEC– pigs (data not shown). A significantly higher proportion of DEX+/PEPEC+ pigs than DEX–/PEPEC+ pigs (P = 0.003) demonstrated foci of intimately adherent bacteria when all the examined intestinal tissues were considered, this difference being most marked in the colon when tissues were considered individually (P = 0.011) (Table 2). Moreover, among the A/E-positive pigs, DEX+/PEPEC+ pigs demonstrated a substantially higher but not significant (P = 0.37 in the ileum and caecum; P = 0.09 in the colon) proportion of ICME with intimately adherent bacteria than DEX–/PEPEC+ pigs (Fig. 2B). In DEX+/PEPEC+ pigs, extensive foci of intimately adherent bacteria were observed; enterocytes beneath intimately adherent bacteria were irregular, condensed, and hypereosinophilic; some were desquamated in the intestinal lumen (Fig. 3A). Inflammation was mild to minimal and limited to colonized areas, being characterized by the presence of PMNs in the subbasal lamina propria and by increased numbers of PMNs in the villous capillaries and venules. No villous atrophy and no histopathological evidence of confounding infections were observed. In contrast, in DEX–/PEPEC+ pigs, only a few scattered A/E lesions with no obvious changes in the morphology of enterocytes beneath adherent bacteria were observed among intestinal sections examined.



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  		FIG. 2. Dexamethasone significantly increases the bacterial colonization of O45-positive Nalr bacteria (P = 0.008) (A) and substantially increases the mean percentage of intercrypt mucosal epithelia with intimately adherent bacteria (ICME+) in the ileum (P = 0.37), cecum (P = 0.37), and colon (P = 0.09) of pigs experimentally challenged with PEPEC strain ECL1001 (B). (B) Extensive foci of intimately adherent bacteria were observed mostly in the ileum and colon of DEX+/PEPEC+ pigs, whereas only a few nonextensive foci of individual intimately adherent bacteria with no obvious change in associated enterocytes were observed in DEX–/PEPEC+ pigs. Results are presented as raw data (A) or as raw data among the A/E-positive pigs (B) with the horizontal bar as the median. A value P of <0.05 was taken to be statistically significant.


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  		TABLE 2. Distribution of foci of intimately-adherent bacteria in intestinal tissues from weaned pigs challenged with the PEPEC strain ECL 1001, following treatment or not with dexamethasone



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  		FIG. 3. Representative HPS-stained ileal section of a DEX+/PEPEC+ pig, showing multiple foci of intimately adherent bacteria and irregular enterocyte morphology (A, arrows). Further observation using rabbit anti-O45 and donkey anti-rabbit Rhodamine Red-X-conjugated antisera confirmed the presence of O45-positive bacteria (B, arrowhead) in foci of intimately adherent bacteria. (C) Multilabeled IFA of an ileal section from a DEX+/PEPEC+ pig, stained with 5 µg ml of DAPI–1 allowed detection of intimately adherent bacteria (blue, arrow), together with red (Rhodamine RedX) and green (fluorescein isothiocyanate) staining, corresponding to colocalized intimin and Tir proteins beneath intimately adherent bacteria, respectively (arrowheads). (D and E) Separate scans of the color channels to allow individual detection of either intimin (D, arrowhead) or Tir (E, arrowhead) on the same intestinal section. Bars, 50 (C), 100 (D and E), or 200 (A and B) µm.

IFA staining revealed O45-positive bacteria in all examined foci of intimately adherent bacteria, corresponding to the challenge PEPEC strain ECL1001 (Fig. 3B). Tissues for which typical intimately adherent bacteria were observed on light microscopy also demonstrated typical A/E lesions with actin condensation and formation of cup-like pedestals on TEM (Fig. 4).



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  		FIG. 4. Representative TEM ileal tissue section of a DEX+/PEPEC+ pig showing extensive, typical A/E lesions with effacement of the microvilli, actin condensation (arrowhead) beneath intimately adhered bacteria, and formation of cup-like pedestals. Bar, 10 µm.

Macroscopic examination of the ileal segments demonstrated that Peyer's patches were highly developed in DEX–/PEPEC+ pigs but were reduced in size in DEX+ pigs, whether challenged or not. The reduction in size of ileal Peyer's patches was also observed in HPS-stained ileal sections (data not shown).

Pigs that received DEX, whether challenged or not with the PEPEC strain, developed mild to moderate diarrhea and also showed truncal obesity, two common characteristics of glucocorticoid excess (42, 44), starting 24 to 48 h after the initial oral intake of DEX. This condition persisted until the end of the experimental procedure. Nevertheless, neither dehydration nor other relevant clinical signs were observed during the experimental procedure for any of the pigs used in this study.

Treatment with dexamethasone resulted in a marked decrease in endogenous blood cortisol levels in weaned pigs. Endogenous blood cortisol levels, which are known to be greatly lowered following high-dose corticosteroid therapy (42), were measured and used as a marker of the presence of a high dose of dexamethasone in the serum of the weaned pigs used in this study. Median blood cortisol levels were significantly lower (P < 0.0001) in DEX+/PEPEC– and DEX+/PEPEC+ (<10.2 nmol/liter, the analytical sensitivity of the test used) pigs than in DEX–/PEPEC– (178 nmol/liter) and DEX–/PEPEC+ (179 nmol/liter) pigs.

Intimin and Tir are both expressed in A/E lesions in vivo in pigs treated with dexamethasone and challenged with PEPEC strain ECL1001. IFA was performed on formalin-fixed, paraffin-embedded tissue sections from infected pigs to investigate the expression of the intimin and its translocated receptor Tir in the foci of intimately adherent bacteria. The expression of intimin, either colocalized with Tir protein or alone, was frequently observed in foci of intimately adherent bacteria found in the ileum, cecum, and colon of DEX+/PEPEC+ pigs, using a multilabeling IFA with anti-ECL1001 intimin and Tir polyclonal antibodies (Fig. 3C to E), whereas Tir protein was only rarely detected in absence of the intimin expression (data not shown). The intimin was localized at the bacterial surface, whereas Tir expression appeared distinct from the bacteria and seemed to be localized inside the epithelial cells, mostly on the apical surface at sites where bacterial intimate adherence occurred (Fig. 3C to E).

PEPEC strain ECL1001 induces an enhanced intestinal production of IL-1ß, IL-6, IL-8, and IL-12p40 proinflammatory cytokines in weaned pigs. The ability of PEPEC strain ECL1001 to induce expression at the transcriptional level, of proinflammatory cytokines IL-1ß, IL-6, IL-8, IL-12p40, and TNF-{alpha}, as well as iNOS, was investigated by semiquantitative RT-PCR in Peyer's patch-containing (PP-containing) or PP-lacking ileum segments of weaned pigs necropsied 96 h after initial bacterial challenge.

IL-6 and IL-8 mRNA levels were significantly higher (P = 0.03 and P = 0.01, respectively) in challenged DEX–/PEPEC+ pigs (n = 5) than in unchallenged DEX–/PEPEC– pigs (n = 5) in PP-containing ileum segments (Fig. 5). On the other hand, IL-1ß mRNA levels were significantly higher (P = 0.023), IL-12p40 mRNA levels were higher although not significantly, and IL-6 mRNA levels were significantly lower (P = 0.035) in challenged DEX–/PEPEC+ pigs than in unchallenged DEX–/PEPEC– pigs in PP-lacking ileum segments (Fig. 5). No statistically significant differences between the DEX–/PEPEC– and DEX–/PEPEC+ pigs were noted for the other tested cytokines, including TNF-{alpha} and iNOS (data not shown) (Fig. 5).



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  		FIG. 5. PEPEC strain ECL1001 induces the upregulation of the mRNA levels of IL-6 (P = 0.03) and IL-8 (P = 0.01) in nontreated, challenged weaned pigs (DEX–/PEPEC+), compared to nontreated, nonchallenged pigs (DEX–/PEPEC–), in Peyer's patch-containing ileum segments (PP-positive, •). On the other hand, it induces the upregulation of the mRNA levels of IL-1ß (P = 0.02) and, to a lesser extent, of IL-12p40 in DEX–/PEPEC+ pigs, compared to DEX–/PEPEC–, and the downregulation (P = 0.03) of the mRNA levels of IL-6 in PP-lacking ileum segments (PP-negative, {square}). Dexamethasone also exerts significant effects on the expression mRNA levels of IL-12p40 (P = 0.005) in DEX+/PEPEC+ pigs, compared to DEX–/PEPEC+ pigs. No other significant modulation of cytokine expression was observed. The DEX+/PEPEC– group represents unchallenged pigs treated with dexamethasone. Results are presented as arbitrary units, and the horizontal bar indicates the median of each group. A P value of <0.05 was taken to be statistically significant.

Dexamethasone blocks the intestinal production of proinflammatory cytokines in weaned pigs challenged with PEPEC strain ECL1001. In spite of the finding that that the levels of mRNA corresponding to some cytokines were modulated following challenge with the PEPEC strain ECL1001, only a few scattered A/E lesions were observed in DEX–/PEPEC+ pigs. Hence, the effect of dexamethasone, which induced a significant increase in the bacterial colonization and prevalence of A/E lesions in challenged DEX+/PEPEC+ pigs, on the intestinal mRNA levels of proinflammatory cytokines and iNOS was investigated in PP-containing or PP-lacking ileum segments of weaned pigs necropsied at 96 h after initial bacterial challenge.

Interestingly, lower levels of mRNA were observed with challenged DEX+/PEPEC+ pigs than with challenged DEX–/PEPEC+ pigs for IL-1ß and IL-12p40 in PP-lacking ileal segments and IL-6 in PP-containing ileal segments. These differences were significant (P = 0.005) for IL-12p40 (Fig. 5). No statistically significant differences were noted for the other tested cytokines (data not shown).


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DISCUSSION
 
We have clearly demonstrated that the host immune status influences the development of attaching and effacing lesions in weaned pigs. Dexamethasone, given orally for 7 days at an immunosuppressive dose, significantly enhanced intestinal PEPEC colonization and development of extensive A/E lesions in weaned pigs experimentally challenged with a PEPEC strain. Hence, in contrast to preliminary challenge experiments carried out in our laboratory using weaned pigs, where no immunosuppressive treatment was given, we have successfully reproduced typical intimate adherence leading to A/E lesions, which were attributable to the challenge strain, as evidenced by the presence of O45+ bacteria in the foci of A/E lesions. We also showed that endogenous blood cortisol levels were significantly lower in DEX+ pigs than in DEX– pigs, strongly suggesting the presence of a high dose exogenous glucocorticoid dexamethasone in the serum of the DEX+ pigs used in this study (42).

This challenge model permitted us to examine the involvement of intimin and its translocated intimin receptor Tir in the development of A/E lesions by PEPEC strain ECL1001 in weaned pigs. Our finding that these proteins are frequently expressed in a colocalized pattern beneath intimately adherent bacteria strongly suggests that these two effectors are virulence factors implicated in the development of A/E lesions by PEPEC in vivo in weaned pigs, strengthening conclusions which have to date been based mostly on experiments carried out under in vitro culture conditions (13, 30). Our results also corroborate the observations for the related murine A/E pathogen C. rodentium (12), where Tir protein is an essential virulence factor needed for actin condensation, intestinal colonization, and colonic hyperplasia in mice.

Effects of glucocorticoids, such as dexamethasone, include inhibition of the secretion of IL-2, granulocyte-macrophage colony-stimulating factor, IL-4, IL-6 (5), gamma interferon (IFN-{gamma}) (25), and TNF-{alpha} (15), and of IL-12p40 by human monocytic cells (33). These effects are mediated via inhibition of various proinflammatory transcription factors such as activator protein-1, nuclear factor of activated T cells, NF-{kappa}ß, and the STAT pathway (1). The importance of T and/or B cells in the development of prolonged inflammatory and crypt hyperplasia associated with C. rodentium-induced A/E lesions in RAG1 knockout mice has recently been demonstrated (69). It has also been hypothesized that the underlying host defense defect resulting in these lesions may be specific to the gastrointestinal tract and possibly involve the intestinal epithelium, since enterocytes are the host cells that primarily interact with C. rodentium (68). Enterocytes are considered as the watchdogs for the natural immune system (16, 41), and there is a growing body of in vivo and in vitro evidence that supports the production of proinflammatory cytokines by enterocytes. For example, rat colonocytes reportedly express IL-1ß mRNA and mouse and human Paneth cells are possible sources of TNF-{alpha}, rabbit M cells reportedly secrete IL-1{alpha}, and human (47) and rat enterocytes possess histochemically detectable IL-6, as well as IL-6 receptors (50, 60). In addition to enterocytes, other cell types participate in the immune surveillance of the mucosal surface. These cells include intestinal dendritic cells (61), intraepithelial lymphocytes and B and T cells in PP, M cells in the dome epithelium (56), and monocytes/macrophages (32).

The need for a fuller understanding of the mucosal immune consequences of PEPEC infection in weaned pigs led us to investigate the proinflammatory response in PP-containing and PP-lacking regions of the ileum, using RT-PCR. Interestingly, a significant upregulation of mRNA levels of proinflammatory cytokines IL-1ß, IL-6, and IL-8 was observed in the ileum of DEX–/PEPEC+ pigs, in which few A/E lesions were observed, but not in pigs that received dexamethasone prior to experimental challenge (DEX+/PEPEC+), in which more extensive A/E lesions were observed. Moreover, comparisons between DEX–/PEPEC+ and DEX+/PEPEC+ pigs confirmed a significant inhibitory effect of dexamethasone on the expression of IL-12p40, as previously described (33). Dexamethasone also tended to inhibit the expression of IL-1ß and IL-6 but to upregulate IL-8. The absence of significant differences between DEX–/PEPEC+ and DEX+/PEPEC+ pigs may be explained by the number of samples tested in our study. The finding of a low cytokine response in challenged DEX+/PEPEC+ pigs in which extensive A/E lesions were observed suggests that this response may contribute to protection of the weaned pig against development of A/E lesions. Our findings are in agreement with the study by Ramirez et al., who showed a stimulation of the expression of proinflammatory cytokines secreted both by enterocytes and by PP lymphocytes which can activate effector mechanisms in the epithelium, in 2-month-old rabbits orally inoculated with rabbit EPEC strain E22 (48). Furthermore, this cytokine response profile, which included IL-6 and IL-1ß and which may be involved in the development of the diarrhea induced by EPEC, depends on the presence intimin. Although our results demonstrated that dexamethasone exerted significant inhibitory effects on the expression of proinflammatory cytokines, it remains possible that this steroid hormone-like molecule (52) could have had some effect on the expression of bacterial virulence genes, such as intimin and Tir, as has been observed for norepinephrine, which participates in the initiation of the quorum sensing mechanism (58). This mechanism has been shown in vitro to control the expression of type III secretion gene transcription and protein secretion in STECO157:H7 and EPEC (57) and in vivo to influence the colonization of the porcine intestine by STECO157:H7 (29). Such an effect on the expression of bacterial virulence genes could have positively influenced the colonization and the further development of A/E lesions by ECL1001 in DEX+/PEPEC+ pigs. Nevertheless, further investigations are required to address such hypothesis.

The differences observed in our study regarding the upregulation of cytokines between the PP-containing and PP-lacking regions of the ileum strongly suggest the involvement of different host immune cells or structures in PEPEC pathogenesis. Indeed in PP, IL-6 may be synthesized and secreted by dome epithelial cells and macrophages and directly target differentiated B cells beneath the PP. IL-6 is a multifunctional cytokine implicated in secretory IgA synthesis by differentiated B cells in the presence of dendritic cells and in initialization of a local acute-phase response through the synthesis of C-reactive protein (23, 34). Moreover, IL-6 is required for a protective immune response to systemic E. coli infection in mice (10). For its part, IL-8 may be synthesized directly by the epithelial cells of the dome epithelium or by macrophages and/or fibroblasts of the lamina propria to initiate neutrophil recruitment (50). This recruitment was found to be mild to minimal on HPS-stained sections examined in this study (data not shown). On the other hand, the upregulation of IL-1ß and, to a lesser extent, IL-12p40 observed in PP-lacking regions of the ileum could be the result of direct stimulation of macrophages by PEPEC, thus allowing the immune response to be oriented as Th1 or Th2.

PP and M cells appear to be involved in the A/E pathogenic process in vitro and in vivo (26, 46). M cells are constantly transporting bacterial products and other bioactive molecules from the lumen into the dome region of the PP (34). Dexamethasone is known to considerably reduce the size of ileal PP and markedly deplete M cells and lymphocytes in the dome epithelium by necrosis and apoptosis, respectively (51). Impaired uptake and transport of PEPEC immune particles to PP via M cells may also have contributed to the increased susceptibility to A/E lesions in DEX+/PEPEC+ pigs by allowing bacteria to evade immune detection at the mucosal level.

In vitro studies have shown that EPEC and STECO157:H7 stimulate early upregulation of the transcription factor NF-{kappa}ß and of the production of IL-8 (22, 53), while prolonged infection resulted in a downregulation of NF-{kappa}ß, IL-6, and IL-8 in an EspB-dependent manner (21). In our study, the upregulation of IL-6 and IL-8 observed with nontreated, challenged weaned pigs may be the result of an A/E lesion-independent stimulation of epithelial cells in the absence of EspB translocation within the host cell cytoplasm, as previously demonstrated by Savkovic et al. with infected T84 intestinal cells (53). On the other hand, the significant downregulation of IL-6 in PP-lacking but not in PP-containing regions of the ileum could reflect prolonged infection of enterocytes (21), although this will need further investigation.

Additionally, Simmons et al. (55) confirmed an important role for IL-12 and IFN-{gamma} in limiting C. rodentium infection to the murine colonic epithelium, hypothesizing that the enhanced susceptibility of gene knockout IL-12- and IFN-{gamma}-deficient mice might be due, in part, to an attenuated expression of the inducible ß-defensin mBD-3. The implication of ß-defensin cannot be discussed with respect to the data reported in our study. However, IL-12p40 seems to play a role in the resistance to bacterial colonization and development of A/E lesions in weaned pigs challenged with PEPEC strain ECL1001, as it appears to be upregulated following infection, and the inhibition of its expression was associated with more extensive A/E lesions in pigs. Another study demonstrated that one of the murine host defense mechanisms encountered by C. rodentium during its infection of the colon is the upregulation of iNOS expression (67). Moreover, in vitro studies demonstrated that pilopolysaccharide of EPEC downregulates nitric oxide synthesis in characterized human small intestinal lamina propria fibroblasts (7). iNOS-derived nitric oxide is a central effector molecule in the innate immune system, and its primary function in host defense appears to be to damage and destroy pathogens (40). In the present study, iNOS was not significantly modulated in any of the intestinal segments tested but nevertheless showed a trend to be upregulated in the ileum, particularly in PP-containing regions.

Taken together, these results strongly suggest that the host immune status influences the development of A/E lesions in weaned pigs. In vivo, it appears that IL-1ß, IL-6, IL-8, and, to a lesser extent, IL-12p40 are expressed during infection of weaned pigs by PEPEC and that these cytokines may contribute to the natural resistance of weaned pigs to PEPEC colonization and to the development of A/E lesions, at least in the ileum. Our results also strengthen the argument that the expression of both intimin and Tir is a relevant mechanism in the pathogenesis of PEPEC infections in vivo. To our knowledge, this is the first report of the involvement of intimin and Tir proteins of any AEEC infections in vivo. As a growing body of evidence now suggests that existing in vitro infection models cannot be extrapolated directly to colonization and disease processes in vivo (12, 37), the model described in this paper will now permit further study of the pathogenesis of EPEC infections in vivo, leading to a fuller understanding of the interactions between the immune system of the host, the bacteria, and their various effectors.


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ACKNOWLEDGMENTS
 
We thank Clarisse Desautels, Éric Dionne, Luc Héroux, Brigitte Lehoux, Jade-Pascale Prévost Lemyre, Joanie Lussier, Chantal Riendeau, Andrée Seyer, and Donald Tremblay for technical assistance; Guy Beauchamp for statistical analysis; and Diane Montpetit for transmission electron microscopy.

This work was supported in part by grants to J.M.F. and J.H. from the Fond Québécois de la Recherche sur la Nature et les Technologies, grant 0214; the Natural Sciences Engineering Research Council of Canada, strategic grant 215841-98 and discovery grant 2294 (J.M.F.); and by EU-Community Quality of Life Project QLK2-2000-00600.


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FOOTNOTES
 
* Corresponding author. Mailing address: Groupe de recherche sur les maladies infectieuses du porc (GREMIP), Faculté de médecine vétérinaire, Université de Montréal, Québec, Canada. Phone: (450) 773-8521, ext. 18342. Fax: (450) 778-8108. E-mail: john.morris.fairbrother{at}umontreal.ca. Back

Editor: A. D. O'Brien


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Infection and Immunity, September 2005, p. 5514-5523, Vol. 73, No. 9
0019-9567/05/$08.00+0     doi:10.1128/IAI.73.9.5514-5523.2005
Copyright © 2005, American Society for Microbiology. All Rights Reserved.



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