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Infection and Immunity, November 2005, p. 7281-7289, Vol. 73, No. 11
0019-9567/05/$08.00+0     doi:10.1128/IAI.73.11.7281-7289.2005
Copyright © 2005, American Society for Microbiology. All Rights Reserved.

Intestinal Innate Immunity to Campylobacter jejuni Results in Induction of Bactericidal Human Beta-Defensins 2 and 3

Matthias Zilbauer,1 Nick Dorrell,2* Parjeet K. Boughan,1 Andrew Harris,3 Brendan W. Wren,2 Nigel J. Klein,1 and Mona Bajaj-Elliott1*

Department of Infectious Diseases and Microbiology, Institute of Child Health, London,1 Department of Infections and Tropical Diseases, London School of Hygiene and Tropical Medicine, London,2 MRC Molecular Pathogenesis Centre for Infectious Disease, Institute for Cell and Molecular Science, Barts' and the London, Queen Mary's School of Medicine and Dentistry, London, United Kingdom3

Received 8 March 2005/ Returned for modification 26 April 2005/ Accepted 8 July 2005


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ABSTRACT
 
Campylobacter jejuni is the most prevalent cause of bacterial diarrhea worldwide. Despite the serious health problems caused by this bacterium, human innate immune responses to C. jejuni infection remain poorly defined. Human ß-defensins, a family of epithelial antimicrobial peptides, are a major component of host innate defense at the gastrointestinal mucosal surface. In this study, the effect of two different C. jejuni wild-type strains on human intestinal epithelial innate responses was investigated. Up-regulation of ß-defensin gene and peptide expression during infection was observed and recombinant ß-defensins were shown to have a direct bactericidal effect against C. jejuni through disruption of cell wall integrity. Further studies using an isogenic capsule-deficient mutant showed that, surprisingly, the absence of the bacterial polysaccharide capsule did not change the innate immune responses induced by C. jejuni or the ability of C. jejuni to survive exposure to recombinant ß-defensins. This study suggests a major role for this family of antimicrobial peptides in the innate immune defense against this human pathogen.


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INTRODUCTION
 
Campylobacter jejuni is the most prevalent cause of bacterial diarrhea in humans and may be responsible for as many as 500 million cases of gastroenteritis worldwide each year (10, 35). The main source of transmission to humans is the consumption or handling of contaminated livestock, especially poultry (25). Poultry such as chickens are frequently highly colonized with C. jejuni (up to 1010 bacteria per g of cecal contents) in an apparently commensal association that causes little or no pathology (33). However, just 100 C. jejuni 81-176 cells are sufficient to cause gastroenteritis in humans (5). Other sources through which C. jejuni enters the food chain include cattle, water, and milk (21, 25). The clinical presentation varies from severe, inflammatory, bloody diarrhea to a generally mild, noninflammatory, watery diarrhea (1). Although the disease is generally mild and rarely requires hospitalization, it is occasionally associated with severe, potentially life-threatening postinfectious complications, such as Guillain-Barré syndrome (26). Given the medical and public health importance of Campylobacter infection, it is remarkable that C. jejuni is one of the least-understood enteropathogens; innate immune responses remain poorly defined.

It is now widely accepted that the gastrointestinal (GI) epithelium not only provides a physical barrier between the lumen and the underlying mucosa but also functions as a critical sensor of infection through the production of an array of cytokines, chemokines, and antimicrobial peptides (4, 18, 27, 28). Studies investigating the role of intestinal epithelial innate defense during C. jejuni infection are very limited. Previous in vitro studies have shown increased epithelial interleukin 8 (IL-8) production during C. jejuni infection (14, 15); however, the exact role of IL-8 remains unclear. Endogenous antimicrobial peptides of the human alpha- and ß-defensin family, LL-37 and lysozyme, are known to be expressed by the GI Paneth cells and epithelia. It has become increasingly clear that these molecules may play a central role in host-microbe cross talk, thus contributing to GI innate defense at the mucosal surface (7, 8, 13, 16, 32). Several members of the human ß-defensin (hBD) family have been identified during GI infection and inflammation (4, 37, 39, 41). hBD-1 is constitutively expressed, suggesting this peptide may play a role in immune surveillance in a healthy host. In contrast, the expression of hBD-2 and hBD-3 is augmented during infection and inflammation (27, 39, 41). To date, no studies have been conducted investigating the regulation and contribution of these defensins to the pathophysiology of C. jejuni infection.

C. jejuni expresses several potential virulence determinants of which the best characterized include flagellum-mediated motility, adhesins, and invasive capability (36). The genome sequence of C. jejuni NCTC11168 revealed the presence of a previously unsuspected capsular polysaccharide (CPS) locus that encodes a structure similar to the group II CPS described for Escherichia coli (19, 20, 29). Encapsulated bacteria are often associated with invasive or otherwise serious infections (31). It was reported recently that CPS protects Klebsiella pneumoniae against the host innate immune defense by limiting the interactions of antimicrobial peptides with the bacterial membrane targets (6). A capsule-deficient C. jejuni 81-176 kpsM mutant demonstrates increased surface hydrophobicity and serum sensitivity and a reduced ability to invade INT407 cells and is also less virulent in a ferret diarrheal disease model (2). The presence of CPS may allow C. jejuni to colonize the intestinal mucus layer, evade phagocytosis, and resist or modulate the host innate immune response.

C. jejuni causes a spectrum of clinical disease; yet in the majority of healthy individuals, the infection is short lived and self limiting, suggesting an important role for the innate immune response in detecting and clearing the bacterium. In this study, we report the effect of C. jejuni infection on intestinal epithelial innate defense gene and peptide expression and the ability of hBDs to act as bactericidal agents against C. jejuni. We also explored the role of the CPS by utilizing an isogenic kpsM mutant in our coculture experiments. Importantly, our in vitro study shows hBDs to be potent bactericidal agents against C. jejuni, suggesting a major role for this family of antimicrobials in host innate defense against this diarrheal agent, possibly via enhanced clearance in a healthy host.


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MATERIALS AND METHODS
 
Bacterial strains and growth conditions. The C. jejuni strains used in this study were the wild-type strains 81-176 (5, 21), a gastroenteritis isolate from a multistate outbreak from contaminated milk; 11168H, a hypermotile isolate that was derived from the source of C. jejuni NCTC11168 used for genome sequencing, which shows high levels of intestinal colonization in a chick colonization model (17); and an isogenic capsule-deficient 11168H kpsM mutant (19). C. jejuni was grown at 37°C in a microaerobic chamber (Don Whitley Scientific, Ltd., Shipley, United Kingdom) containing 85% N2, 5% O2, and 10% CO2 on blood agar plates, consisting of Columbia agar base (Oxoid, Basingstoke, United Kingdom) supplemented with 7% (vol/vol) defibrinated horse blood (TCS Microbiology, Botolph Claydon, United Kingdom) or in Mueller-Hinton broth (Oxoid). Both mid-log-phase bacteria grown for 24 h in Mueller-Hinton broth as well as "mixed-phase" bacteria grown on blood agar plates for 24 h were used for bacterium-epithelial cell coculture experiments and antimicrobial assays.

Interaction of C. jejuni with intestinal epithelial cells. All reagents for tissue culture, RNA extraction, and reverse transcription-PCR were obtained from Invitrogen (Paisley, United Kingdom) unless stated otherwise. The human intestinal epithelial cell lines Caco-2 and HT-29 were cultured in Dulbecco's modified essential medium DMEM plus GlutaMAX supplemented with 10% (vol/vol) fetal calf serum (Sigma-Aldrich, Gillingham, United Kingdom), 100-U/ml penicillin, 100-µg/ml streptomycin, and 1% nonessential amino acids and maintained at 37°C in 5% CO2 and 95% air. For coculture experiments, cells were grown in a 25-cm2 tissue culture flask to >90% confluence and maintained in serum and antibiotic-free medium overnight prior to coculture with 108 CFU/ml (multiplicity of infection = 100) of C. jejuni strains for 6, 10, and 24 h. Each infection was terminated by removal of the supernatant and washing the cells twice with phosphate-buffered saline (PBS). IL-1ß stimulation was routinely included as a positive control, since the cytokine is a known potent agonist of ß-defensin expression (23, 27, 41). Cells and supernatants were frozen at –80°C until required or processed immediately for total RNA or protein extraction.

RNA extraction and reverse transcription-PCR analysis. Total RNA was isolated using a monophasic solution of phenol and guanidine thiocyanate (TRIZOL), followed by chloroform extraction and isopropanol precipitation. Total RNA was quantified by spectrophotometry, and 5 µg total RNA was reversed transcribed to cDNA at 42°C with 1 µg of oligo(dT) primer (Amershan-Pharmacia, St. Albans, United Kingdom), 1 mM (each) deoxynucleotide triphosphates, and Moloney murine leukemia virus reverse transcriptase in a volume of 20 µl, following the manufacturer's protocol. A total of 5 µl (1 µl for the housekeeping gene glyceraldehyde-3-phosphate dehydrogenase [GAPDH]) of cDNA was routinely amplified with 20 pmol of each oligonucleotide primer (Sigma-Aldrich), 1.5 mM MgCl2, 200 µM deoxynucleoside triphosphates, and 0.5 U of Taq polymerase. Primer sequences were as described previously (3, 12). After a 3-min hot start at 94°C, the amplification profile used was denaturation at 94°C for 90 s, annealing at 60°C for 90 s, and extension at 72°C for 90 s. PCRs were allowed to continue for 34 to 37 cycles, ensuring termination in the linear phase of the reaction. The PCR products were analyzed on 2% (wt/vol) agarose gels.

Western blotting. To confirm a parallel increase in hBD-2 and hBD-3 peptides in response to C. jejuni infection, Western blotting was performed. Briefly, protein from control uninfected and 24-h-infected Caco-2 cell supernatants and lysates was extracted overnight in 20% acetic acid prior to centrifugation (1,200 x g; 20 min) and lypholization. Protein was resuspended in 10 mM acetic acid and quantified by Bio-Rad protein (Bradford) assay. A total of 600 µg of total protein was dissolved directly in Tris-Tricine loading buffer (Bio-Rad Laboratories, Hemel Hampstead, United Kingdom) and subjected to 16% Tris-Tricine sodium dodecyl sulfate-polyacrylamide gel electrophoresis. After electrophoresis, gels were transferred onto a polyvinylidene difluoride membrane (Amersham Biosciences, St. Albans, United Kingdom) at 0.8 mA/cm2 for 40 min. Nonspecific binding was blocked in 5% nonfat milk-Tris-buffered saline for 1 h. After overnight incubation with the primary antibodies (1:500 dilution for hBD-2 [Santa Cruz Biotechnology, Calne, United Kingdom]; and 1:1,000 for hBD-3 [Gentaur Molecular Products, Brussels, Belgium]), the membrane was washed three times with Tris-buffered saline-Tween, followed by incubation with horseradish peroxidase-conjugated rabbit anti-goat immunoglobulin G and goat anti-rabbit immunoglobulin G (1:1,000; Dako, Ltd., High Wycombe, United Kingdom), respectively. The reaction was developed by the enhanced chemiluminescent method (Amersham Biosciences, Amersham, United Kingdom).

Antimicrobial assay. Recombinant ß-defensin peptides (Peprotech, Ltd., London, United Kingdom) were reconstituted in 10 mM acetic acid according to the supplier's instructions. C. jejuni strains were cultured as described above and suspended in hypotonic buffer (10 mM phosphate buffer, 50 mM NaCl) (13). Approximately 105 CFU/ml organisms were exposed to recombinant ß-defensins for 30 min at 37°C under microaerobic conditions. The experiments were terminated by plating serial dilutions of the reaction mixture in triplicate onto blood agar; viable bacteria were counted after 3 days. Bactericidal activity was calculated as a percentage of colony counts of bacteria not exposed to antimicrobial peptides but subjected to the same experimental conditions. Results from the antimicrobial assay represent the mean of three independent experiments.

Scanning electron microscopy (SEM). Silicon wafers were immersed in 0.1% (vol/vol) poly-L-lysine (Sigma-Aldrich) for 15 min and allowed to air dry before 100 µl of bacterial suspension was adsorbed onto a wafer for 15 min. Wafers coated with bacteria were submerged in 0.5% (vol/vol) glutaraldehyde (Sigma-Aldrich) in PBS for 5 min at ambient temperature and stored in PBS at 4°C until further processing. The wafers were washed in distilled water prior to immersion in 1% (wt/vol) osmium tetroxide (Sigma-Aldrich) for 15 min. Following a second series of three washes in distilled water, the wafers were passed through a series of ascending concentrations of ethanol washes (30%, 50%, 70%, and 90%) for 5 min each and then two washes in absolute ethanol for 10 min. The wafers were then dried, with absolute ethanol as the transfer fluid, in a Balzer's CPD 030 critical point dryer (Bal-Tec, Liechtenstein). The wafers were finally mounted on carbon supports, sputter coated with gold using a Balzer's SCD 030 sputter coater (Bal-Tec), and examined with a JEM 1200EXII scanning transmission electron microscope (Jeol, Tokyo, Japan) operating in the scanning mode (40 to 60 kV).

Transient transfection and luciferase reporter assay. For promoter-reporter studies, Caco-2 and HT-29 cells were seeded in a 96-well plate at a density of 2 x 104 cells per well in 200 µl DMEM and used for transfection at 60 to 80% confluence. For transfection, FuGENE reagent (Roche, Lewes, United Kingdom) and Opti-MEM (Invitrogen, Paisley, United Kingdom) were mixed and incubated at room temperature for a minimum of 15 min. DNA was added at a ratio of 4:1 (DNA in micrograms: FuGene in microliters) as recommended by the manufacturer. The amount of total DNA (230 ng) transfected was equalized among experiments by the addition of appropriate amounts of empty vector plasmid (pcDNA; Stratagene, Cambridge, United Kingdom). Test plasmids encoding full-length hBD-2, hBD-3 (kindly provided by S. J. Ong, Institute of Child Health, London, United Kingdom), IL-8, and NF-{kappa}B (kindly provided by A. G. Bowie, Trinity College, Dublin, Ireland) promoter-luciferase constructs (60 ng/well) were cotransfected with a Renilla luciferase construct (20 ng/well), the latter used to account for cell loss and transfection efficiency. The mixture was added directly to cultured 96-well plates and incubated at 37°C for 24 h. For stimulation experiments, medium was replaced by 100 µl of DMEM containing C. jejuni (108 CFU/ml) or IL-1ß (20 ng/ml). After incubation at 37°C for 20 h, cells were washed with 200 µl PBS, and 50 µl passive lysis buffer (Promega, Southampton, United Kingdom) was added prior to detection. Firefly and Renilla luciferase activity was measured with a 96-well plate luminometer (Lucy 1; Anthos Biotech, Salzburg, Austria).

Statistics. Results are presented as means ± standard error of the mean and are consistent with the results of two to three separate experiments, which were performed in triplicate. Statistical analyses were via an unpaired, two-tailed, t test.


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RESULTS
 
Wild-type C. jejuni strains induce ß-defensin gene and peptide expression in intestinal epithelium cells. It has been shown previously that C. jejuni strain 81-176 induces expression of IL-8 in INT407 intestinal epithelial cells (14, 15). IL-8, hBD-1, hBD-2, and hBD-3 gene expression during infection with C. jejuni wild-type strains 81-176 and 11168H was investigated in two different intestinal epithelium (Caco-2 and HT-29) cell lines (Fig. 1a and b). Both C. jejuni strains induced IL-8 gene expression, in agreement with previously published reports. Expression of hBD-1 in uninfected cells was observed, and this basal level did not change during infection of both cell lines, indicating that the presence of C. jejuni does not affect the constitutive expression of hBD-1. No hBD-2 and hBD-3 gene expression was observed in uninfected control cells; however, both C. jejuni strains induced the expression of these two antimicrobial peptides in both the cell lines. Induction of IL-8, hBD-2, and hBD-3 was time dependent, with the highest level of mRNA expression achieved 10 h postinfection (Fig. 1a and b). hBD-2 and hBD-3 gene expression was maintained up to the 24-h time point. Interestingly, the induction of hBD-3 expression was consistently modest in HT29 cells, compared to that in the Caco-2 cell line.



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  		FIG. 1. Modulation of human ß-defensin and IL-8 gene expression during C. jejuni infection in Caco-2 (a) and HT-29 (b) cells. (a) hBD-1 was expressed constitutively and no further modulation was observed during the course of infection. In contrast, IL-8, hBD-2, and hBD-3 mRNA levels were up-regulated during infection with both wild-type (WT) strains. Modulation of all innate genes tested was similar for both strains with no significant difference in kinetics or magnitude. Cells at 8 h post-IL-1ß stimulation were also analyzed for IL-8 and ß-defensin expression in parallel and treated as a positive control to the live infection. (b) Findings were reconfirmed with HT-29 cells. hBD-3 expression in this cell line was minimal.

To determine whether increased expression of hBD-2 and hBD-3 mRNA correlated with elevated levels of the corresponding peptide, culture supernatants and cell lysates from control and 24-h postinfected samples were analyzed by Western blotting. hBD-2 and hBD-3 peptide was undetectable in the control supernatant and cellular lysate extract (Fig. 2). In contrast, immunoreactive hBD-2 and hBD-3 were detected in the infected culture supernatant and cell lysate, respectively. The presence of secreted hBD-2 in the supernatant compared to increased hBD-3 peptide in cell lysate suggests differential kinetics of peptide secretion.



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  		FIG. 2. hBD-2 and hBD-3 peptides are induced in response to C. jejuni 11168H. Control and infected Caco-2 cell supernatants and lysates were subjected to Tris-Tricine sodium dodecyl sulfate-polyacrylamide gel electrophoresis, followed by Western blotting. Both peptides were undetectable in unstimulated control cells. hBD-2 peptide was detected in supernatant (top); in contrast, higher levels of hBD-3 peptide were observed in the cell lysates of infected cells (bottom). Recombinant peptides were included as a positive control.

Recombinant hBD-2 and hBD-3 exhibit potent bactericidal activity against C. jejuni. Our in vitro studies (Fig. 1 and 2) showed modulation of hBD-2 and hBD-3 gene and peptide expression during C. jejuni infection. If this induction reflects events in vivo, the peptides may play a role in limiting colonization and infection by exhibiting bactericidal activity against the bacterium. In preliminary experiments, we investigated whether bacterial growth phase influenced susceptibility to ß-defensins. For this purpose, we compared susceptibility of mid-log-phase C. jejuni 11168H against that of a "mixed-phase" culture and found after 30-min incubation that both hBD-2 and hBD-3 at a concentration of 10–6 M exhibited equal potency against either bacterial culture system (Fig. 3a). Interestingly, hBD-1 was less potent and showed greater variability between experiments. The reason for this remains unclear. Further, dose-dependent studies utilizing a mixed-phase culture system were performed. At a concentration between 10–6 M and 10–7 M, both inducible hBD-2 and hBD-3 were bactericidal, rendering >99% of the bacteria nonviable (Fig. 3b). At lower concentrations, both peptides were less active. Further studies of hBD-3 killing were performed to investigate the kinetics of its bactericidal activity. Within 5 min of exposure to hBD-3, >99% killing was achieved, suggesting that the bactericidal activity is not only potent but also very rapid (data not shown).



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  		FIG. 3. Bactericidal activity of recombinant ß-defensins against C. jejuni 11168H. (a) 10–6 M ß-defensins were added to plate-grown (mixed-culture) and broth-grown (mid-log-phase) 11168H and incubated at 37°C for 30 min prior to plating. The control sample used was bacteria resuspended in 10 mM phosphate buffer in the absence of antimicrobial peptide and subjected to the same experimental conditions. (b) Dose-dependent bactericidal activity of hBD-2 and hBD-3 against 11168H.

hBD-3 causes severe damage to C. jejuni cell membrane. The mechanism(s) by which hBDs cause damage to bacteria remains unclear. Using SEM, the structural damage caused to C. jejuni 11168H by hBD-3 was investigated. Bacteria incubated in buffer alone over the experimental time (30 min) were undamaged, retaining their spiral shape (Fig. 4a) and found to be viable when plated onto solid medium. In contrast, bacteria incubated with hBD-3 were nonviable when plated onto solid medium and showed apparent thinning out and/or peeling of the cell wall with subsequent loss of cytoplasmic contents (Fig. 4b to d).



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  		FIG. 4. C. jejuni 11168H was incubated with recombinant hBD-3 and examined by scanning electron microscopy. (a) Bacteria incubated in buffer alone were undamaged, retaining their spiral shape and found to be viable when plated onto solid medium. In contrast, bacteria incubated with hBD-3 were nonviable when plated onto solid medium and showed apparent thinning out and/or peeling of the cell wall (b), with formation of membrane-enclosed blebs (c and d) and subsequent loss of cytoplasmic contents.

C. jejuni CPS is not protective against the antimicrobial action of hBD-2 and hBD-3. It has been reported that capsule polysaccharide mediates bacterial resistance to host antimicrobial peptides (6). To determine whether the C. jejuni CPS provides protection against the bactericidal activity of ß-defensins, the ability of a capsule-deficient isogenic 11168H kpsM mutant to survive exposure to hBDs was investigated. The kpsM mutant proved to be equally susceptible to the antimicrobial action of hBD-2 and hBD-3, with >99% bacterial cells nonviable after 30 min of exposure (Fig. 5). As noted for the wild type-strain, the kpsM mutant was susceptible to hBD-2 and hBD-3 at a 10–7 M peptide concentration and higher, with complete loss of ß-defensin activity at 10–8 M (data not shown). Interestingly, hBD-1 showed higher levels of bactericidal activity against the kpsM mutant than the wild-type strain (Fig. 5). However, this increase in bactericidal activity could not be confirmed statistically, due to the high variation in hBD-1 activity.



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  		FIG. 5. Comparison of the antimicrobial activity of recombinant hBDs (10–6 M) versus C. jejuni 11168H and the isogenic kpsM mutant. Both inducible ß-defensins proved to be very potent, with >99% kill after 30-min incubation. In contrast, the constitutively expressed hBD-1 exhibited a significantly lower level of antimicrobial activity. No significant difference was observed between 11168H and the kpsM mutant for all hBDs tested. WT, wild type.

C. jejuni CPS plays a minimal role in modulating intestinal epithelial innate gene expression. The C. jejuni CPS has been shown to have an important role in virulence; an 81-176 kpsM mutant exhibited a reduced ability to invade INT407 cells and was found to be less virulent in a ferret diarrheal disease model (2). To investigate the potential role of CPS in modulating host defenses at the site of infection, Caco-2 and HT-29 cells were infected with the 11168H kpsM mutant and levels of IL-8, hBD-1, hBD-2, and hBD-3 gene expression were investigated. Even in the absence of the CPS, the kpsM mutant was able to induce expression of IL-8, hBD-2, and hBD-3; the magnitude and the time dependency of the response appeared similar to those observed for the wild-type strain. Further, no difference in induction profiles was noted between the two cell-lines (Fig. 6a and b). In view of previous reports, this observation was surprising. To confirm these findings and for indication of differences in levels of gene expression, a transient transfection promoter-luciferase reporter assay was employed. At present little is known about intestinal epithelial cell signaling events following C. jejuni infection. The transcription factor NF-{kappa}B is known to play a major role in IL-8 and hBD-2 gene regulation (9, 27), and C. jejuni has been shown to activate NF-{kappa}B in intestinal epithelial cells (24). The degree of activation of NF-{kappa}B, IL-8, hBD-2, and hBD-3 occurring during infection with the wild-type 11168H and the kpsM mutant was investigated. A two- to threefold increase in NF-{kappa}B, IL-8, and hBD-2 promoter activity was observed with Caco-2 cells (Fig. 7a to c). In the same experiments, IL-1ß, a known potent agonist for IL-8 and hBD-2 gene expression, showed a fourfold induction. The hBD-3 promoter does not have any potential NF-{kappa}B binding sites. The similar induction of hBD-3 by both the wild type and the kpsM mutant (Fig. 7d) suggests the activation of other signaling pathways during C. jejuni infection. These studies confirmed the ability of the kpsM mutant to modulate epithelial innate immunity to a degree similar to that observed for the wild-type strain.



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  		FIG. 6. Modulation of hBD and IL-8 gene expression by C. jejuni 11168H kpsM strain in Caco-2 (a) and HT-29 (b) cells. The capsule-deficient mutant exhibits a similar pattern in modulating innate immune gene expression as the wild-type strain (Fig. 1). hBD-1 was expressed constitutively, while hBD-2, hBD-3, and IL-8 were up-regulated in both cell lines. con, uninfected controls.



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  		FIG. 7. Caco-2 cells were transiently transfected with luciferase reporter vectors containing IL-8 (a), NF-{kappa}B (b), hBD-2 (c), or hBD-3 (d) promoters. Luciferase activity was assessed as a ratio between firefly and Renilla luciferase. Data are expressed as the n-fold increase in luciferase activity compared to that in unstimulated cells. Error bars indicate the standard error of the mean of values obtained from two to three independent experiments performed in triplicate. Differences between luciferase activity levels were analyzed by Student's t test. Significant up-regulation of all promoters tested was observed (P < 0.01). No significant difference was found between 11168H and the isogenic kpsM mutant (P = 0.5 ± 0.1).


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DISCUSSION
 
There is accumulating evidence that host epithelial antimicrobial peptides contribute to host defense at mucosal surfaces (8, 11, 32). Dynamic modulation of ß-defensins has been demonstrated with various models of GI infection and inflammation (16, 27, 41). C. jejuni is a commensal organism of poultry but represents a major health burden in humans. Poultry are routinely colonized with up to 1010 C. jejuni cells per g of cecal contents in an apparently commensal association that causes little or no pathology (33). In contrast, as few as 100 C. jejuni cells are sufficient to cause severe inflammatory gastroenteritis in humans in volunteer studies (5). The bacterium causes a spectrum of clinical disease; yet in the majority of healthy individuals, the infection is short lived and self limiting, suggesting an important role for innate immunity in detecting and clearing the infective agent.

The aim of this study was to investigate the potential role and regulation of intestinal epithelial ß-defensins during C. jejuni infection. This is the first report suggesting dynamic modulation of hBD-2 and hBD-3 gene and peptide expression by wild-type C. jejuni. Our in vitro studies showed rapid (within 6 h) induction of all three innate immune genes (IL-8, hBD-2, and hBD-3) tested. The magnitude of this response differed for the two wild-type strains tested. This may reflect differences in the ability of the bacterium to invade intestinal epithelial cells, as 81-176 is reported to have a greater invasive potential than the NCTC11168 strain (30). It has previously been shown that different C. jejuni strains are reported to induce different levels of IL-8 secretion and that this is directly related to their invasive potential (14). Uninfected intestinal epithelial cells did not express hBD-2 or hBD-3, but the presence of C. jejuni resulted in increased expression over the 24-h experimental time period. This suggests a directed innate immune response against this intestinal pathogen.

We further confirmed that the increase in hBD-2 and hBD-3 mRNA correlated with increased peptide expression. Interestingly, hBD-2 peptide was recovered from culture supernatant; in comparison, greater expression of hBD-3 was found in the cellular fraction. A similar differential distribution of hBD-2 and hBD-3 was recently reported in a skin model of infection and inflammation (34).

The observed potent bactericidal properties of ß-defensins against C. jejuni suggest that these peptides may play a role in limiting infection in vivo. The constitutively expressed hBD-1 was also bactericidal against C. jejuni, albeit with less potency than hBD-2 and hBD-3. It is possible that hBD-1 action may be sufficient in certain circumstances to prevent bacterial colonization from proceeding. In this scenario, synergy among constitutively expressed antimicrobials present in the vicinity may play an important role (40). Alpha-defensins, LL-37, and lysozyme may synergize with hBD-1 in vivo to prevent successful adhesion by C. jejuni to the intestinal epithelial cell surface. However, if C. jejuni successfully adheres to and colonizes the intestinal surface and starts to invade individual epithelial cells, the inducible hBD-2 and hBD-3 may come into effect. One may hypothesize that the differential compartmentalization of hBD-2 and hBD-3 as suggested by Western blotting also contributes to host defense, such that bacteria that adhere to the intestinal epithelia are likely to be more susceptible to secretory hBD-2. In comparison, bacteria that successfully invade the epithelia may encounter the killing capacity of hBD-3. Although the concentrations of hBD-2 and hBD-3 achieved in the intestinal mucosa during C. jejuni infection are unknown, the present bactericidal dose-dependent studies are compatible with those suggested for hBD-2 for gram-negative bacteria (22). We propose that the overall net effect of greater killing capacity of the inducible ß-defensins contributes to the self-limiting nature of C. jejuni infection in a healthy individual.

Bacterial CPS has been reported to play a role in modulating host immune responses and is known to provide resistance against host antimicrobial peptides in other pathogens (6). The investigation of the role of C. jejuni CPS in eliciting intestinal epithelial responses surprisingly showed that a capsule-deficient isogenic kpsM mutant exhibited an ability to modulate IL-8 and ß-defensin gene expression that was similar to that of the 11168H wild-type strain. To date, two mechanisms involving bacterial adherence and/or invasion and the presence of C. jejuni cytolethal distending toxin (CDT) have been implicated in IL-8 induction (15). An 81-176 kpsM mutant has previously been shown to have a reduced ability to invade INT407 cells (2). This suggests that if the 11168H kpsM mutant employed in our study also has reduced ability to invade intestinal epithelial cells, then induction of IL-8 by this mutant may occur primarily via the CDT-dependent mechanism. Further studies are required to confirm the role of CDT in eliciting IL-8 and ß-defensin gene expression during C. jejuni infection.

Equal potency exhibited by the recombinant ß-defensins towards the capsule-deficient mutant is also surprising. It was predicted that the CPS surrounding C. jejuni would be protective against the peptides' bactericidal activities, as occurs with K. pneumoniae (6). One reason why no protective effect of C. jejuni CPS was observed in the present investigation may be due to the rapid bactericidal activity of hBD-2 and hBD-3, as >99% of bacterial cells were nonviable within 5 min of exposure to 10–6 M hBD-3. We therefore conducted experiments at lower peptide concentrations to observe any increased susceptibility of the capsule mutant to hBD-2 or hBD-3. We found no difference in hBD-3 bactericidal activity against the wild type and the kpsM mutant when tested between concentration ranges of 5 x 10–8 M and 10–6 M. In the case of hBD-2, slight differences in survival between the wild type and the isogenic mutant were observed, but this difference was statistically not significant. Both peptides, however, were completely ineffective against either organism at 10–8 M. There is some evidence that C. jejuni down-regulates CPS as the bacteria adheres to and invades intestinal epithelial cells (N. Dorrell and P. H. Everest, unpublished data). This is consistent with our findings suggesting that C. jejuni CPS may not be involved in modulating intestinal epithelial innate immune responses.

No studies have been reported investigating the regulation and contribution of the human ß-defensins to the pathophysiology of C. jejuni infection. In this study, evidence is presented showing that C. jejuni modulates ß-defensin gene expression and that these peptides are potent bactericidal agents. Host genetic variation that results in low levels of hBD-2 and hBD-3 induction may result in a defective antimicrobial barrier function at the mucosal surface, thus predisposing such individuals towards infection. There is evidence that this is indeed the case in patients suffering from Crohn's disease (37, 38). Intestinal epithelial innate defense is likely to play a critical role in not only sensing the presence of C. jejuni but also actively killing the bacteria, possibly directly resulting in a self-limiting disease. In a broader context, the induction of these ß-defensins may have an important role in maintaining a healthy GI tract.


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ACKNOWLEDGMENTS
 
We thank Keith Pell (School of Biological Sciences, Queen Mary, University of London) for his assistance with scanning electron microscopy and Abdi Elmi (London School of Hygiene and Tropical Medicine) for technical support.

Matthias Zilbauer was funded by scholarships from the Innovative Medizinische Forschung (IMF) Muenster, Germany, and the Medical Research Council (MRC, United Kingdom).


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FOOTNOTES
 
* Corresponding author. Mailing address for Mona Bajaj-Elliott: Department of Infectious Diseases and Microbiology, Institute of Child Health, London WC1N 1EH, United Kingdom. Phone: 44-20-7907-2215. Fax: 44-20-7813-8494. E-mail: m.bajaj-elliott{at}ich.ucl.ac.uk. Mailing address for Nick Dorrell: Department of Infectious and Tropical Diseases, London School of Hygiene and Tropical Medicine, London WC1E 7HT, United Kingdom. Phone: 44-20-7927-2838. Fax: 44-20-7637-4314. E-mail: nick.dorrell{at}lshtm.ac.uk. Back

Editor: V. J. DiRita


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Infection and Immunity, November 2005, p. 7281-7289, Vol. 73, No. 11
0019-9567/05/$08.00+0     doi:10.1128/IAI.73.11.7281-7289.2005
Copyright © 2005, American Society for Microbiology. All Rights Reserved.



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