Modulation of Host Immune Responses by the Cytolethal Distending ...

INFECTION AND IMMUNITY, Aug. 2006, p. 4496–4504 0019-9567/06/$08.00⫹0 doi:10.1128/IAI.00503-06 Copyright © 2006, American Society for Microbiology. All Rights Reserved.

Vol. 74, No. 8

Modulation of Host Immune Responses by the Cytolethal Distending Toxin of Helicobacter hepaticus Jason S. Pratt,1,2 Kacey L. Sachen,1 Heather D. Wood,1 Kathryn A. Eaton,4,5 and Vincent B. Young1,2,3* Department of Microbiology and Molecular Genetics,1 National Food Safety and Toxicology Center,2 and Infectious Diseases Unit, Department of Internal Medicine,3 Michigan State University, East Lansing, Michigan 48824, and Unit for Laboratory Animal Medicine4 and Department of Microbiology and Immunology,5 University of Michigan School of Medicine, Ann Arbor, Michigan 48109 Received 28 March 2006/Returned for modification 3 May 2006/Accepted 11 May 2006

Persistent murine infection with Helicobacter hepaticus leads to chronic gastrointestinal inflammation and neoplasia in susceptible strains. To determine the role of the virulence factor cytolethal distending toxin (CDT) in the pathogenesis of this organism, interleukin-10-deficient (IL-10ⴚ/ⴚ) mice were experimentally infected with wild-type H. hepaticus and a CDT-deficient isogenic mutant. Both wild-type H. hepaticus and the CDTdeficient mutant successfully colonized IL-10ⴚ/ⴚ mice, and they reached similar tissue levels by 6 weeks after infection. Only animals infected with wild-type type H. hepaticus developed significant typhlocolitis. However, by 4 months after infection, the CDT-deficient mutant was no longer detectable in IL-10ⴚ/ⴚ mice, whereas wild-type H. hepaticus persisted for the 8-month duration of the experiment. Animals infected with wild-type H. hepaticus exhibited severe typhlocolitis at 8 months after infection, while animals originally challenged with the CDT-deficient mutant had minimal cecal inflammation at this time point. In follow-up experiments, animals that cleared infection with the CDT-deficient mutant were protected from rechallenge with either mutant or wild-type H. hepaticus. Animals infected with wild-type H. hepaticus developed serum immunoglobulin G1 (IgG1) and IgG2c responses against H. hepaticus, while animals challenged with the CDT-deficient mutant developed significantly lower IgG2c responses and failed to mount IgG1 responses against H. hepaticus. These results suggest that CDT plays a key immunomodulatory role that allows persistence of H. hepaticus and that in IL-10ⴚ/ⴚ mice this alteration of the host immune response results in the development of colitis. Campylobacter species, certain Escherichia coli strains, Shigella dysenteriae, Haemophilus ducreyi, and Actinobacillus actinomycetemcomitans (reviewed in references 24, 35, and 36). The active subunit of CDT, CdtB, has structural and functional homology to mammalian DNase I (8, 23, 32). It has been proposed that this DNase activity is responsible for the cell cycle arrest that is a key feature of the CDT-mediated cytopathic effect in vitro (7, 17, 20, 33). The role of CDT in the in vivo pathogenesis of organisms that elaborate this toxin has been investigated. Fox and colleagues demonstrated that wild-type C. jejuni, but not an isogenic mutant lacking CDT, triggered gastroenteritis in NF-␬Bdeficient mice (16). The same group recently reported that CDT expression by H. hepaticus is required for long-term colonization of outbred Swiss Webster mice (18). We recently reported that an isogenic H. hepaticus mutant that lacked CDT production was able to colonize C57BL/6 interleukin-10-deficient (IL-10⫺/⫺) mice, but colonization with the CDT-deficient strain was associated with a significant reduction in IBD activity 6 weeks after infection compared to that in animals infected with wild-type H. hepaticus (53). These results suggest that CDT expression may represent a bacterial adaptation that influences the interaction between the bacterium and the host immune system. Therefore, to determine more precisely the role of CDT in the modulation of the host response to H. hepaticus, multiple infection studies were carried out with C57BL/6 IL-10⫺/⫺ mice challenged with wild-type H. hepaticus and a CDT-deficient isogenic mutant.

Helicobacter species are responsible for chronic human and veterinary infections (44). In humans, H. pylori infection can last for decades, associated with a subclinical gastritis. Longterm infection with H. pylori can lead to the development of neoplastic disease, including gastric cancer and mucosa-associated lymphoid tissue lymphomas (37). In addition to H. pylori and other gastric Helicobacter species, the enterohepatic Helicobacter species (EHS) have emerged as veterinary and human pathogens also associated with long-term infection and the development of neoplastic disease (13, 44). The EHS H. hepaticus was originally discovered as the causative agent for the development of chronic hepatitis and hepatocellular cancer in A/JCr mice (15, 46). It was subsequently determined that H. hepaticus infection in mice with altered immune function was also associated with the development of a condition that mimicked human inflammatory bowel disease (IBD) (3, 5, 22). Long-term infection with H. hepaticus in animals that develop IBD can lead to the development of colon cancer (9, 10, 28). H. hepaticus and a number of other EHS have been shown to produce a cytotoxin that is a member of the cytolethal distending toxin (CDT) family (4, 52, 54). CDT is a tripartite bacterial toxin that is encountered in a number of pathogenic gramnegative organisms, including Campylobacter jejuni and other * Corresponding author. Mailing address: Room B42, Food Safety and Toxicology Building, Michigan State University, East Lansing, MI 48824. Phone: (517) 432-3100. Fax: (517) 432-2310. E-mail: youngvi @msu.edu. 4496

H. HEPATICUS CDT AND IMMUNITY

VOL. 74, 2006 TABLE 1. Design of infection studies No. of animals in study (sacrifice, days postchallenge) Challenge strain

Wild type (3B1) 3B1::Tn20 (CDT negative) None (uninfected)

6-wk time Longitudinal Longitudinal Rechallenge course (2, study 1 study 2 study (35) 4, 8, 14, 42) (251) (225)

15 15

10 10

10 40

15

10

10

4 6

MATERIALS AND METHODS Bacterial strains and cell lines. The wild-type H. hepaticus strain 3B1 (the type strain, ATCC 51488) was obtained from the American Type Culture Collection (Manassas, VA). The isogenic mutant 3B1::Tn20 was generated by transposon shuttle mutagenesis with allelic exchange into H. hepaticus (53). 3B1::Tn20 has a transposon inserted near the start of cdtA and no longer produces cytolethal distending toxin (53). Wild-type H. hepaticus and the CDT-deficient isogenic mutant strain were grown at 37°C for 3 to 4 days in a microaerobic environment, which was maintained in vented GasPak jars without a catalyst after evacuation to ⫺20 mm Hg and equilibration with a gas mixture consisting of 80% N2, 10% CO2, and 10% H2. H. hepaticus was grown on tryptic soy agar (TSA) supplemented with 5% sheep blood and with 20 ␮g/ml chloramphenicol (all from Sigma, St. Louis, MO) for the chloramphenicol-resistant transposon mutant. Animals. All animal protocols were reviewed and approved by the Michigan State University All University Committee on Animal Use and Care. Breeding pairs of Helicobacter-free C57BL/6 IL-10⫺/⫺ mice were obtained from the Jackson Laboratories (Bar Harbor, ME) and housed with autoclaved food, bedding, and water, with cage changes performed in a laminar flow hood. For infection studies, 4- to 6-week-old C57BL/6 IL-10⫺/⫺ mice were transferred to the University Research Containment Facility at Michigan State University and housed under the same conditions as the breeding pairs. Animals were housed in groups of up to five animals per microisolator cage (Table 1). Murine infection with H. hepaticus. H. hepaticus was harvested after 48 h of growth on agar plates and resuspended in a small volume of tryptic soy broth. The optical density (OD) at 600 nm of the inoculum was measured and 10-fold serial dilutions of the inoculum plated to quantify the CFU used for infection. Mice were inoculated with a single dose of a suspension of bacteria with an OD of 1.0 at 600 nm (⬃1 ⫻ 108 CFU) in a volume of 0.2 to 0.3 ml. Bacteria were introduced directly into the stomach with a 24-gauge ball-tipped gavage needle. Control mice were inoculated with sterile tryptic soy broth. Detection of H. hepaticus in mouse feces and tissues. Fecal pellets from the animals in one cage were collected and pooled to monitor colonization status. Culture for H. hepaticus was accomplished by homogenizing feces in 0.5 ␮l of phosphate-buffered saline and plating 50 ␮l on TSA supplemented with 5% sheep blood, 20 ␮g/ml cefoperazone, 10 ␮g/ml vancomycin, and 2 ␮g/ml amphotericin B. DNA was isolated from fecal pellets as described previously (47). Briefly, fecal pellets were homogenized, and following a low-speed centrifugation to remove large particulate matter, DNA was isolated using a commercial kit (QIAGEN tissue kit; QIAGEN, Valencia, CA). DNA was isolated from gastrointestinal tissue collected at the time of necropsy by using the QIAGEN tissue kit. Direct, single-stage PCR amplification to detect H. hepaticus was performed using the primers 5⬘ GCA TTT GAA ACT GTT ACT CTG 3⬘ (B38) and 5⬘ CTG TTT TCA AGC TCC CC 3⬘ (B39), which produce a 417-bp amplicon (42). PCR was performed using 5 ml of template at approximately 100 ng/␮l of DNA extracted from fecal or tissue samples. Each 25-␮l PCR mixture contained 20 pmol of each primer, 200 ␮M of each deoxynucleoside triphosphate, and 1.5 U of Taq DNA polymerase in a final concentration of 10 mM Tris-HCl, 50 mM KCl, and 1.5 mM MgCl2 (Ready To Go PCR beads; Amersham Pharmacia Biotech, Piscataway, NJ). Cycling conditions included 30 cycles of 30 seconds at 94°C, 45 seconds at 54°C, and 45 seconds at 72°C. PCR products were visualized by agarose gel electrophoresis. To provide a measure of relative levels of fecal shedding of H. hepaticus, a nested PCR system was developed. The first-stage PCR employs primers 5⬘ GCT ATG ACG GGT ATC C 3⬘ (C97) and 5⬘ ACT TCA CCC CAG TCG CTG 3⬘ (C05), which amplify the small-subunit rRNA genes from all known Helicobacter species (14), yielding an approximately 1,200-bp amplicon. PCR was performed

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using Ready To Go PCR beads. Cycling conditions included 30 cycles of 30 seconds at 94°C, 60 seconds at 58°C, and 90 seconds at 72°C. The nested stage was performed using primers B38 and B39 (described above) with 1 ␮l of the reaction mixture from the primary PCR as the template. PCR products were visualized by agarose gel electrophoresis. In our experience, this nested PCR has sufficient sensitivity to detect 25 to 50 genome equivalents of H. hepaticus in a volume of 1 ␮l (approximately 100 ng) of DNA purified from mouse feces. Mouse necropsy and histologic procedures. Mice were euthanatized by CO2 asphyxiation. Collection of the ileocecocolic junction and preparation of hematoxylin-and-eosin-stained sections for histopathologic examination were performed as described previously (53). Histologic sections were examined using the following scoring system for inflammation: 0, normal; 1, small multifocal lamina proprial and/or transepithelial leukocyte accumulations; 2, coalescing mucosal inflammation with or without early submucosal extension; 3, coalescing mucosal inflammation with prominent multifocal submucosal extension with or without follicle formation; and 4, severe diffuse inflammation of mucosa, submucosa, and deeper layers. T-RFLP analysis. Terminal restriction fragment length polymorphism (TRFLP) analysis was performed as detailed previously (21). Briefly, PCR amplification employing primers targeting bacterial 16S rRNA genes (8F and 1492R (41) was performed on each DNA sample. The 8F primer was linked to the fluorescent dye 6-carboxyfluorescein (Integrated DNA Technologies, Coralville, IA), and the 1492R primer was unlabeled. The PCR products were purified using GFX purification columns (Amersham Pharmacia Biotech). Two hundred nanograms of purified PCR amplicon was cut individually with the restriction enzymes HhaI and MspI (New England Biolabs, Beverly, MA) for 1 to 2 h at 37°C (27). The DNA fragments were separated on an ABI 3100 Genetic Analyzer automated sequence analyzer (Applied Biosystems Instruments, Foster City, CA) in GeneScan mode. The 5⬘ terminal restriction fragments (TRFs) were detected by excitation of the 6-carboxyfluorescein molecule attached to the forward primer. The sizes and abundances of the fragments were calculated using GeneScan 3.7. T-RFLP profiles were analyzed as follows. To standardize each profile for the quantity of labeled DNA present in each sample, the sum of TRF peak heights in each profile being compared was calculated. The sum of peak heights generally varied less than twofold over all of the profiles. Each sum of TRF peak heights was normalized to the lowest sum of peak heights of the comparison samples. This yielded a correction factor that was applied to each peak in a given profile. The resultant peak heights were filtered to eliminate peaks with a height below the noise threshold (set at a relative fluorescence value of 50). The fraction of the total signal provided by the H. hepaticus TRF was determined by dividing the peak height of the H. hepaticus TRF by the sum of the total peak height in a given profile. Preparation of H. hepaticus antigens. H. hepaticus was cultured on TSA blood plates and suspended in phosphate-buffered saline at an OD at 600 nm of 1.5 to 3.0. The suspension was then frozen at ⫺70°C, thawed, and pelleted at 5,000 rpm for 10 min. The pellet was resuspended in B-PER I reagent (Pierce Chemical Co., Rockford, IL) and then centrifuged at 13,000 rpm for 5 min. The protein concentration of the supernatant was measured by the Lowry technique (BioRad protein assay; Bio-Rad Laboratories, Hercules, CA). Aliquots of the supernatant were stored at ⫺70°C. Evaluation of serum antibody responses to H. hepaticus. At necropsy, blood was collected via cardiac puncture from CO2-asphyxiated mice. For a subset of mice, blood was collected every 3 weeks during the course infection by saphenous vein puncture. Blood was centrifuged at 10,000 rpm for 10 min, and the serum was preserved with 15 mM sodium azide. An enzyme-linked immunosorbent assay (ELISA) was used to detect H. hepaticus-specific serum immunoglobulin G (IgG) and IgA. Nunc Polysorb 96-well plates (Nalge Nunc International, Rochester, NY) were coated with 100 ml of a 10-mg/ml concentration of H. hepaticus protein in carbonate buffer (pH 9.6) overnight at 4°C. Plates were blocked with 1% bovine serum albumin in phosphate-buffered saline. Serum was diluted 1/100, and biotinylated secondary antibodies included goat anti-mouse IgG1 (Sigma, St. Louis, MO) diluted 1/5,000 and goat anti-mouse IgG2c (Southern Biotech, Birmingham, AL) diluted 1/1,000. Incubation with extravidin-peroxidase (Sigma) at a dilution of 1/1,000 was followed by incubation with 2,2⬘-azine-di(3-ethylbenzthiazoline sulfonate) (ABTS) horseradish peroxidase substrate (Pierce) for color development. Optical density at 405 nm was recorded by an ELISA plate reader (VERSAmax; Molecular Devices, Sunnyvale, CA). Statistical analysis. Statistical analysis was performed using the JMP statistical package (SAS, Cary, NC). Categorical inflammation scores were compared by the nonparametric Wilcoxon rank sum test with statistical significance set to a P value of ⬍0.05. ELISA data were analyzed using a one-way analysis of variance (ANOVA), and the Tukey-Kramer honestly significant difference test was used

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FIG. 1. Categorical inflammation scores following the temporal development of typhlocolitis in IL-10⫺/⫺ mice infected with wild-type H. hepaticus or a CDT-deficient isogenic mutant. Animals infected with wild-type H. hepaticus developed severe inflammation within 8 days after experimental challenge, whereas animals infected with the CDTdeficient mutant did not develop significant colitis.

to identify groups with significantly different means with an alpha level set to 0.05.

RESULTS An isogenic CDT-deficient H. hepaticus mutant demonstrates delayed colonization kinetics and triggers minimal typhlocolitis. We have previously shown that infection of IL10⫺/⫺ mice with a CDT-deficient H. hepaticus mutant is associated with decreased IBD activity 6 weeks after challenge compared to that in animals infected with wild-type H. hepaticus (53). Both the CDT-deficient mutant and wild-type H. hepaticus were detected by culture and PCR in the feces and tissue of all animals at the end of this 6-week infection study. In order to follow the early colonization kinetics and the development of typhlocolitis in H. hepaticus-infected C57BL/6 IL-10⫺/⫺ mice, a time course infection study was performed. The H. hepaticus mutant 3B1::Tn20 is deficient in CDT production due to a transposon insertion in the cdtA gene of the type strain of H. hepaticus (53). Groups of three mice were challenged via oral gavage with either wild-type H. hepaticus or the isogenic CDT-deficient mutant (Table 1). Animals were sacrificed at 2, 4, 8, 14, and 42 days after experimental challenge and the cecal tissue harvested to assess the development of inflammation and to determine relative levels of colonization of the mucosa by H. hepaticus. Animals infected with the CDT-deficient mutant 3B1::Tn20 did not exhibit significant cecal inflammation at any time after challenge (Fig. 1). Animals infected with wild-type H. hepaticus, however, developed histologically significant typhlocolitis as early as 8 days after challenge (Fig. 1). Uninfected control animals did not develop any significant inflammation (data not shown). Although all animals challenged with either wild-type H. hepaticus or the CDT-deficient mutant had the organism detectable by culture or H. hepaticus-specific PCR of feces, we

FIG. 2. Monitoring of the colonization of IL-10⫺/⫺ mice with H. hepaticus. T-RFLP analysis was used to determine the fraction of the mucosa-associated microbiota represented by H. hepaticus in animals infected with the wild type or the CDT-deficient mutant. The CDTdeficient mutant colonizes with slightly delayed kinetics compared to the wild type but by 42 days after infection reaches a similar level among the cecal microbiota.

wished to compare the colonizations of the cecal mucosa by each isogenic strain. We have previously reported that the 16S rRNA gene-encoding sequence-based technique of T-RFLP analysis can be used to temporally follow the colonization of the cecal mucosa of wild-type mice by wild-type H. hepaticus (21). We used T-RFLP analysis to compare the colonization kinetics of wild-type H. hepaticus and the CDT-deficient mutant in the IL-10⫺/⫺ mice. Wild-type H. hepaticus rapidly colonized the cecal mucosae of IL-10⫺/⫺ mice, comprising approximately 50% of mucosa-associated microbiota by 14 days postchallenge (Fig. 2). These kinetics are similar to those for the colonization of the cecal mucosae of wild-type mice as reported previously (21). The CDT-deficient mutant appeared to have somewhat delayed kinetics, not being detectable as a major TRF at 8 days after challenge and not becoming the dominant TRF until 42 days after challenge. However, at 42 days, the CDT-deficient mutant was the dominant component of the mucosa-associated microbiota in two of the three mice and was easily detectable in the remaining animal (Fig. 2). Thus, the CDT-deficient mutant can still become a significant member of the cecal-associated microbiota, but it does so with delayed kinetics compared to wild type and does not trigger significant typhlocolitis. An isogenic CDT-deficient H. hepaticus mutant exhibits a long-term colonization defect. To determine the role of CDT in the development of long-term sequelae of H. hepaticus infection, additional infection studies were initiated using the isogenic CDT mutant. Four- to 6-week-old C57BL/6 IL-10⫺/⫺ mice were infected with wild-type H. hepaticus strain 3B1 and the CDT-deficient isogenic mutant 3B1::Tn20. An equal num-


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TABLE 2. Chronological monitoring of colonization of IL-10⫺/⫺ mice with H. hepaticus by H. hepaticus-specific PCR and culture Challenge strain

Wild type 3B1::Tn20 (CDT negative) None (uninfected)

PCR result (culture result) at day p.i.: 9

32

45

61

115

251

⫹ ⫹

⫹ (⫹) ⫹ (⫹)

⫹ (⫹) ⫹ (⫹)

⫹ ⫹

⫹ (⫹) ⫺ (⫺)

⫹ (⫹) ⫺ (⫺)

⫺

⫺ (⫺)

⫺ (⫺)

⫺

⫺ (⫺)

⫺ (⫺)

ber of control mice were sham infected with sterile culture broth. There were 10 animals in each experimental group, for a total of 30 animals in this study (Table 1). Animals in all experimental groups were monitored for H. hepaticus colonization by H. hepaticus-specific PCR amplification of DNA isolated from fresh fecal pellets (42) and, at certain time points, by selective culture for H. hepaticus from feces. Since mice are coprophagic, we used the “cage” as the unit of infection when assessing colonization. Thus, there were two cages in each experimental group, and at all times, the results were concordant for each of the two cages for each group. C57BL/6 IL-10⫺/⫺ mice were initially colonized by wild-type H. hepaticus 3B1 and the isogenic CDT-deficient mutant (Table 2). Animals challenged with wild-type H. hepaticus remained colonized for the entire duration of each experiment. Conversely, although the CDT-deficient mutant 3B1::Tn20 was detectable at 61 days postinfection (p.i.) in IL-10⫺/⫺ animals, this mutant was not found by PCR or culture at 115 days p.i. These mice remained negative for H. hepaticus for the remainder of the experiment. At all times when colonization was assessed by both culture and PCR for H. hepaticus, there was concordance between the two methods (Table 2). Mice that clear infection with a CDT-deficient H. hepaticus mutant exhibit minimal typhlocolitis. Histologic examination of the ileocecocolic junctions of the IL-10⫺/⫺ mice challenged with wild-type 3B1 and the isogenic mutant was performed at 251 days postinfection. Of the 30 animals in this experiment, one had to be sacrificed prior to this time. One animal infected with wild-type 3B1 developed rectal prolapse at 130 days p.i. Nine of the 10 control animals had no or minimal evidence of inflammation at the ileocecocolic junction at the time of necropsy (Fig. 3A). One of the control animals had significant inflammation and hyperplasia (Fig. 4). Feces and tissue from this animal were examined carefully for H. hepaticus infection by culture and PCR, and H. hepaticus was not detected in this outlier. Animals with infected with wild-type H. hepaticus exhibited moderate to severe chronic typhlocolitis (Fig. 3B). These animals developed marked mucosal hyperplasia with infiltration of the lamina propria with mononuclear inflammatory cells. This disease was significantly different from findings for uninfected controls (Fig. 4). IL-10⫺/⫺ mice that were initially infected with the CDT-deficient mutant 3B1::Tn20 but eventually cleared the infection at between 61 and 115 days p.i. had minimal signs of inflammation. Two animals had no visible inflammatory infiltrates, while the majority of the remaining animals only had small, scattered aggregates of chronic inflammatory cells with minimal hyperplasia (Fig. 3C). The scores of the animals infected with the CDT-deficient mutant 3B1::Tn20

FIG. 3. Histolopathologic lesions in the cecae of IL-10⫺/⫺ mice 8 months after challenge with isogenic strains of H. hepaticus. IL-10⫺/⫺ mice were sham infected (A) or infected with wild-type H. hepaticus strain 3B1 (B) or the CDT-negative mutant 3B1::Tn20 (C). Mice challenged with wild-type H. hepaticus developed moderate to severe inflammation and hyperplasia. Mice challenged with the CDT-deficient mutant, which was cleared by 4 months after infection, exhibited minimal disease at 8 months.

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FIG. 5. Monitoring of the loss of colonization with a CDT-deficient H. hepaticus mutant by using a nested PCR assay. H. hepaticus could be detected in the feces of animals challenged with wild-type (WT) H. hepaticus for the entire 225-day duration of the experiment. There was progressive loss of colonization with the CDT-deficient mutant starting 75 days after infection as judged by the primary stage of the PCR assay and starting at day 125 as judged by the nested assay.

FIG. 4. Categorical inflammation scores for uninfected IL-10⫺/⫺ mice and mice infected with wild-type H. hepaticus strain 3B1 and the CDT-deficient isogenic mutant strains 8 months after challenge. Comparisons between groups were performed using the Wilcoxon rank sum test.

were not significantly different from the scores of uninfected controls (Fig. 4) at 8 months postinfection. Monitoring of progressive clearance of a CDT-deficient H. hepaticus mutant. Given the above results, we wished to follow the clearance of the CDT-deficient H. hepaticus mutant from the gastrointestinal tract. To achieve this, an additional, longitudinal study was performed (Table 1). Groups of C57BL/6 IL-10⫺/⫺ mice were infected with wild-type H. hepaticus and the isogenic CDT-deficient mutant 3B1::Tn20. Animals were housed in groups of three to five animals per cage. A total of 40 animals in 11 cages were infected with 3B1::Tn20. Ten animals, divided into four cages, remained as uninfected controls, and an additional 10 animals (also in four cages) were infected with wild-type H. hepaticus strain 3B1. Once again, we used the “cage” as the unit of infection, and the colonization status was assessed using pooled fecal samples taken approximately every 3 weeks from each cage of animals. To provide a measure of the relative levels of colonization, a nested PCR assay was used to monitor colonization. An initial PCR amplification was performed with a pair of primers that amplify the small-subunit rRNA genes of all known Helicobacter species (14), followed by amplification with a nested pair of primers specific for H. hepaticus (42), which were used to monitor colonization in the initial experiment described above. In our hands, this nested PCR assay has the ability to detect between

25 and 50 genome equivalents of H. hepaticus DNA in 1 ␮l (approximately 100 ng) of fecal DNA (data not shown). As before, C57BL/6 IL-10⫺/⫺ mice were initially colonized by wild-type H. hepaticus 3B1 and 3B1::Tn20. Wild-type H. hepaticus colonized the mice for the entire duration of each experiment (Fig. 5). Conversely, although the CDT-deficient mutant 3B1::Tn20 was detectable by the primary PCR in all cages of infected mice sampled at 75 days p.i. in IL-10⫺/⫺ animals, an increasing fraction of the cages were negative by the primary PCR assay. All cages of 3B1::Tn20-infected animals remained positive by the more sensitive nested PCR assay until 117 days postinfection, but then cages became negative by this nested assay with kinetics similar to those seen with the primary PCR assay (Fig. 5). H. hepaticus-specific humoral and cellular immune responses in IL-10ⴚ/ⴚ mice infected with wild-type or CDTdeficient bacteria. It has been demonstrated that mice infected with H. hepaticus develop a significant H. hepaticus-specific antibody response (47). To measure antibody responses against H. hepaticus in infected animals, an ELISA was developed based on a previously published assay (48). Antigens were extracted from plate-grown H. hepaticus via the use of a nonanionic detergent mixture (B-PER; Pierce Chemical Co., Rockford, IL) and used to coat 96-well microtiter plates. The sera from IL-10⫺/⫺ animals in the longitudinal follow-up study were used in this ELISA to monitor the development of an anti-H. hepaticus humoral immune response in animals infected with wild-type H. hepaticus and the CDT-deficient mutant 3B1::Tn20. Thirty animals in the experiment (10 controls, 10 challenged with wild-type H. hepaticus, and 10 challenged with the CDT-deficient mutant) had serum samples taken every 3 weeks, allowing us to follow the development of H. hepaticus-specific humoral immune responses. None of the sera from uninfected C57BL/6 IL-10⫺/⫺ animals contained antibody reactive to H. hepaticus antigens in ELISA (Fig. 6). All animals infected with wild-type H. hepati-

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FIG. 6. Development of H. hepaticus-specific humoral immune responses in IL-10⫺/⫺ animals infected with wild-type and CDT-deficient H. hepaticus. (Top) Animals infected with the wild type and the CDTdeficient mutant developed significant IgG2c responses by 34 days after challenge, but the magnitude of the response was greater in animals infected with wild-type H. hepaticus. (Bottom) Significant IgG1 responses were seen only in animals infected with the wild type. Animals infected with the CDT-deficient mutant did not develop IgG1 responses greater than those in negative controls. Each data point represents the mean OD values for 10 animals, with standard deviations depicted by error bars. ⴱ, P ⬍ 0.05 compared to control mice; §, P ⬍ 0.05 compared to CDT-deficient mice. All mean comparisons were performed by ANOVA and the Tukey-Kramer test.

cus or the CDT-deficient mutant developed IgG2c H. hepaticus-specific humoral immune responses by 34 days after infection (Fig. 6, top panel). However, the magnitude of the responses was significantly greater (P ⬍ 0.05 by ANOVA and the Tukey-Kramer honestly significant difference test at 34 days and all subsequent time points) in the animals that were challenged with wild-type H. hepaticus. This differential response was even more dramatic when H. hepaticus-specific IgG1 responses were measured. Animals challenged with wildtype H. hepaticus developed robust anti-H. hepaticus IgG1 responses by 34 days after infection, but the responses in animals challenged with the CDT-deficient mutant were not significantly different from those in uninfected controls at any time following infection (Fig. 6, bottom panel). At the end of the experiment (225 days after initial infection), analysis of sera from all mice revealed that these differ-


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ences in response were sustained. Animals challenged with wild-type H. hepaticus had average IgG2c responses of 2.12 ⫾ 0.36 (mean ⫾ standard deviation) and average IgG1 responses of 0.91 ⫾ 0.71. This is in contrast to the responses of animals challenged with the CDT-deficient mutant, which had average IgG2c responses of 0.88 ⫾ 0.58 and IgG1 responses of 0.12 ⫾ 0.072. Mice that clear infection with a CDT-deficient H. hepaticus mutant are protected from rechallenge. Once we determined that mice could clear infection with the CDT-deficient H. hepaticus transposon mutant, we wished to determine if clearance was associated with the development of protective immunity. Three cages of IL-10⫺/⫺ mice that were infected with the CDT-deficient mutant 3B1::Tn20 and had cleared infection (as judged by the nested PCR assay) were rechallenged with H. hepaticus. Two cages of mice (a total of six animals, three in each cage) were rechallenged with 3B1::Tn20, and one cage of mice (four animals) was rechallenged with wild-type H. hepaticus 3B1. Colonization status was monitored by the nested PCR assay described above. One cage of mice rechallenged with 3B1::Tn20 did not have H. hepaticus detectable by either the primary or nested PCR assay at 7 and 21 days postinfection. The other cage of three mice rechallenged with 3B1::Tn20 was positive by both primary and nested PCR on day 7 but only by nested PCR on day 21. The cage of animals rechallenged with wild-type H. hepaticus was positive only by the nested PCR assay on both days 7 and 21 postinfection. Since the PCR assays were performed on a pooled fecal sample from each cage, at 35 days after infection, an individual fecal pellet was taken from each animal for PCR analysis of colonization. Of the four mice rechallenged with wild-type H. hepaticus, three had cleared the organism by 35 days after rechallenge. One of the animals was still shedding relatively large numbers of H. hepaticus organisms, since a fecal pellet from this animal was positive by both the primary and nested PCR assays. H. hepaticus was cultured from the feces of this animal and was confirmed to be the wild-type H. hepaticus used for rechallenge and not the CDT-deficient mutant originally used to challenge this animal. Of the animals rechallenged with 3B1::Tn20, two of the six animals were negative for H. hepaticus by PCR. Overall, 5 of 10 animals rechallenged with H. hepaticus were not infected with the organism at 35 days after challenge (P ⫽ 0.0015 by Fisher’s exact test, compared to the animals challenged initially with wild-type H. hepaticus in this experiment). DISCUSSION Chronicity is a key feature of Helicobacter infections. In some cases, this is of minimal significance to the host. However, chronic Helicobacter infection and associated long-term inflammation can lead to the development of neoplastic disease. It is estimated that at least half of the world’s population is chronically infected with H. pylori. Although only a minority of chronically infected individuals go on to develop gastric adenocarcinoma, the sheer magnitude of chronic infections makes this the second leading cause of cancer-related death worldwide (37). Bacteria that are able to cause persistent infections have

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developed varied strategies to evade immune surveillance (29, 39, 50). H. pylori produces the virulence factor vacuolating cytotoxin A (VacA), which has been theorized to allow H. pylori to evade immune surveillance and chronically colonize the gastric mucosa (30). VacA has been shown to inhibit T-cell activation and thus may interfere with the generation of a protective immune response by the host (2, 19). H. hepaticus does not possess a homologue of vacA, the structural gene for VacA. Cytolethal distending toxin, which has not been demonstrated to be present in H. pylori, may serve in a role parallel to that of VacA as an immunoregulatory toxin in H. hepaticus. CDT has been shown to induce apoptosis in primary human peripheral blood mononuclear cells and cultured T-cell lines (34, 40, 43). In addition to a direct effect on T cells, CDT may be able to interfere with immune responses by interfering with antigen-presenting cells. It was recently shown that primary human macrophages and dendritic cells treated with CDT were deficient in cytokine production and in the stimulation of T-cell proliferation (49). In the current study, we provide evidence that CDT is necessary for long-term colonization of the murine gut by H. hepaticus. A CDT-deficient isogenic mutant of H. hepaticus can maintain colonization of IL-10⫺/⫺ mice for at least 2 months but is subsequently cleared by 4 months after experimental infection. This implies that IL-10⫺/⫺ mice infected with the CDT-deficient mutant are able to mount an immune response that results in clearance of the organism. Mice infected with either wild-type H. hepaticus or the CDTdeficient mutant develop significant H. hepaticus-specific humoral immune responses as measured by ELISA. It has been previously reported that mice experimentally challenged with wild-type H. hepaticus develop robust anti-H. hepaticus antibody titers, despite being unable to clear the infection (48). We demonstrate here that IL-10⫺/⫺ mice infected with wild-type H. hepaticus develop greater humoral responses than animals infected with a CDT-deficient mutant. This diminished humoral immune response cannot be explained by lower levels of tissue colonization by the CDT-deficient mutant. T-RFLP analysis revealed that the CDT-deficient mutant has slightly delayed colonization kinetics of the mucosa but is able to reach levels comparable to those reached by wild-type H. hepaticus. Thus, the decreased levels of H. hepaticus-specific antibody seen in animals challenged with the CDT-deficient mutant suggest that CDT expression modulates the host response to the organism. Additional support for the role of CDT in modulating host immune responses comes from the observation that IL-10⫺/⫺ mice infected with the CDT-deficient mutant develop significantly less colonic inflammation than IL-10⫺/⫺ animals infected with wild-type bacteria. In the present study, we demonstrate that over the first 6 weeks following experimental infection and at 8 months after infection, minimal inflammation is seen in mice challenged with a CDT-deficient mutant. The degree of inflammation in animals challenged with the CDT-deficient mutant was not different from that seen in uninfected, control animals. This is in marked contrast to the case for IL-10⫺/⫺ animals infected with wild-type H. hepaticus, where significant colitis was seen as early as 8 days after infection. It is important to note that one of the 20 control animals in the two long-term studies presented here did develop sig-

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nificant typhlocolitis (Fig. 2). This animal was carefully examined for evidence of H. hepaticus infection, and this was not found by culture and PCR of fecal specimens and tissue specimens. We have noted the generation of “spontaneous” colitis in ⱕ5% of the non-H. hepaticus-infected mice in our breeding colony during the 3 years that it has been in existence, particularly among older animals. This emphasizes that although H. hepaticus is sufficient to trigger significant colitis in virtually all experimentally infected IL-10⫺/⫺ animals, it is not a necessary factor. Other, presumably noninfectious triggers, such as mechanical mucosal injury from materials in the feed, can initiate an uncontrolled inflammatory response in these colitis-prone animals, albeit at a much lower rate than H. hepaticus infection. Our results are in general agreement with those of Ge and colleagues, who reported that an independent CDT-deficient H. hepaticus mutant was unable to maintain persistent colonization of outbred Swiss Webster mice (18). In their study, mice infected with either wild-type H. hepaticus or the CDT-deficient mutant developed significant H. hepaticus-specific ELISA reactivity compared to uninfected controls. They also noted that mice infected with the CDT-deficient mutant had decreased H. hepaticus-specific ELISA reactivity compared to animals infected with the wild type. As Ge and colleagues assessed ELISA reactivity only at the termination of their experiment, the lower levels of H. hepaticus-specific antibody in animals challenged with their CDT-deficient mutant may have reflected the time that passed between clearance of the organism and the collection of serum at the end of the experiment. However, our data regarding the longitudinal development of anti-H. hepaticus antibody reactivity in animals suggests that this is a reflection of the differential immune response to wild-type H. hepaticus and a CDT-deficient mutant. As a final demonstration of the differences in the immune responses to wild-type H. hepaticus and a CDT-deficient mutant, we show that animals that clear infection with the CDTdeficient mutant are provided with a degree of protection against reinfection. Initial infection with the CDT-deficient mutant 3B1::Tn20 is associated, in a subset of individuals, with the development of a protective immune response against rechallenge with either 3B1::Tn20 or wild-type H. hepaticus. Although the number of animals rechallenged with H. hepaticus following clearance of the CDT-deficient mutant was small, the development of protective immunity in half of the rechallenged animals was statistically significant. Animals infected with wild-type H. hepaticus (either experimentally or via vertical transmission) remain colonized with the organism for their entire life span. “Spontaneous” loss of colonization does not occur, and antibiotic treatment directed at H. hepaticus is characterized by inconsistent efficacy in eliminating carriage (6). It is formally possible that 3B1::Tn20 has a defect (perhaps unrelated to lack of CDT) that is responsible for the long-term colonization defect. However, the fact that animals that have lost colonization with 3B1::Tn20 rapidly clear rechallenge with wild-type H. hepaticus provides much stronger evidence that this is due to the generation of protective immunity. Therefore, although mice infected with either wild-type H. hepaticus or the CDT-deficient mutant develop H. hepaticus-specific humoral immune responses, there is a significant qualitative (as

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well as quantitative) difference in the immune response, since protective immunity does not develop in animals infected with wild-type organisms. It remains to be determined if the actual H. hepaticus antigens/epitopes that are targeted by the H. hepaticus-specific immune responses differ and if this explains the development of protective immunity in animals infected with the CDT-deficient mutant. The results from the current study suggest that CDT production by H. hepaticus represents a bacterial adaptation that allows long-term persistence within the mammalian host. CDT expression serves to modify the development of host immunity such that the resulting H. hepaticus-specific immune responses fail to clear the organism. In a host with an altered immune system, such as an IL-10⫺/⫺ mouse, this modulation of the H. hepaticus-specific immune response results in the development of dysregulated immunity and colitis. Although the development of severe typhlocolitis in IL-10⫺/⫺ mice following H. hepaticus infection appears to be dependent on CDT production by the bacterium, the resulting disease could be considered to be a reflection of the underlying host defect rather than enhanced “virulence” of CDT-producing H. hepaticus. This line of reasoning can help explain the results obtained by other investigators who have examined the role of CDT in the pathogenesis of bacterial infections. CDT does not appear to play a significant role in the acute pathogenesis of H. ducreyi. Isogenic H. ducreyi CDT mutants were found to be fully virulent in human volunteers and in the temperature-dependent rabbit model of chancroid (26, 51). However, these models examine only the acute state of infection, up to 14 days in the human volunteers and 7 days in the rabbit. However, chancroid ulceration due to H. ducreyi infection is a chronic condition, persisting for months in the absence of effective antimicrobial therapy (25). The authors of these studies speculated that CDT may have a role in the chronic phase of chancroid but not in the acute phase (45). CDT expression is also found in most strains of C. jejuni (11, 12, 38). Isogenic CDT-deficient C. jejuni strains are unable to maintain long-term gastrointestinal colonization of wild-type mice, again suggesting a role for CDT in the escape of immune surveillance (16). These CDT-deficient C. jejuni strains also caused less severe chronic gastrointestinal disease (gastritis and duodenitis) in NF-␬B-deficient mice (16). However, the role for CDT in the acute pathogenesis of Campylobacterassociated human disease is not clear. CDT-negative C. jejuni strains have been found to be associated with human enteric disease, causing some investigators to question the role of CDT in the pathogenic process (1). These same authors demonstrated that an isogenic CDT-deficient C. jejuni strain was able to colonize newborn chicks for up to 5 days, but they did not examine the ability of this mutant to persist long term. Interestingly, they also reported that sera from patients with campylobacteriosis developed neutralizing anti-CDT antibodies, but similar neutralizing antibodies were not found in chickens experimentally infected with CDT-producing C. jejuni (1). The results of experiments with H. hepaticus and C. jejuni suggest that it is not entirely accurate to consider CDT a strict virulence factor for the Epsilonproteobacteria, at least not in the sense of being central to the development of acute bacterially mediated pathology. It might be more appropriate to consider CDT an evolutionary adaptation that plays a key role in the


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establishment of symbiotic relationships between these bacteria and natural eukaryotic hosts. In the case of H. hepaticus, which is widespread in wild and laboratory rodents and thus can be considered to be part of the indigenous murine gastrointestinal microbiota (44), CDT is a bacterial adaptation that allows the bacterium to occupy this environmental niche as a commensal. Intriguing evidence for this hypothesis has been derived from studies that suggest that CDT may play a key role in a symbiotic system involving insects, viruses, and bacteria (31). In summary, we confirm that cytolethal distending toxin has an essential role in maintaining long-term intestinal colonization by H. hepaticus via the modulation of host immune responses. It will be interesting to determine if there are similar roles for CDT in other bacteria that elaborate this toxin. ACKNOWLEDGMENTS We thank Jennifer Cortez for expert technical assistance. This work was supported by a USDA National Needs Fellowship to J.S.P., a Michigan State University Intramural Research Grant Program (IRGP) New Investigator Award to V.B.Y., and a Crohn’s and Colitis Foundation of America Senior Investigator Award to V.B.Y. Additional support was provided by the Michigan State University Center for Microbial Pathogenesis. REFERENCES 1. Abuoun, M., G. Manning, S. A. Cawthraw, A. Ridley, I. H. Ahmed, T. M. Wassenaar, and D. G. Newell. 2005. Cytolethal distending toxin (CDT)negative Campylobacter jejuni strains and anti-CDT neutralizing antibodies are induced during human infection but not during colonization in chickens. Infect. Immun. 73:3053–3062. 2. Boncristiano, M., S. R. Paccani, S. Barone, C. Ulivieri, L. Patrussi, D. Ilver, A. Amedei, M. M. D’Elios, J. L. Telford, and C. T. Baldari. 2003. The Helicobacter pylori vacuolating toxin inhibits T cell activation by two independent mechanisms. J. Exp. Med. 198:1887–1897. 3. Cahill, R. J., C. J. Foltz, J. G. Fox, C. A. Dangler, F. Powrie, and D. B. Schauer. 1997. Inflammatory bowel disease: an immunity-mediated condition triggered by bacterial infection with Helicobacter hepaticus. Infect. Immun. 65:3126–3131. 4. Chien, C. C., N. S. Taylor, Z. Ge, D. B. Schauer, V. B. Young, and J. G. Fox. 2000. Identification of cdtB homologues and cytolethal distending toxin activity in enterohepatic Helicobacter spp. J. Med. Microbiol. 49:525–534. 5. Chin, E. Y., C. A. Dangler, J. G. Fox, and D. B. Schauer. 2000. Helicobacter hepaticus infection triggers inflammatory bowel disease in T cell receptor alpha/beta mutant mice. Comp. Med. 50:586–594. 6. Duysen, E. G., D. L. Fry, and O. Lockridge. 2002. Early weaning and culling eradicated Helicobacter hepaticus from an acetylcholinesterase knockout 129S6/SvEvTac mouse colony. Comp. Med. 52:461–466. 7. Elwell, C., K. Chao, K. Patel, and L. Dreyfus. 2001. Escherichia coli CdtB mediates cytolethal distending toxin cell cycle arrest. Infect. Immun. 69: 3418–3422. 8. Elwell, C. A., and L. A. Dreyfus. 2000. DNase I homologous residues in CdtB are critical for cytolethal distending toxin-mediated cell cycle arrest. Mol. Microbiol. 37:952–963. 9. Engle, S. J., I. Ormsby, S. Pawlowski, G. P. Boivin, J. Croft, E. Balish, and T. Doetschman. 2002. Elimination of colon cancer in germ-free transforming growth factor beta 1-deficient mice. Cancer Res. 62:6362–6366. 10. Erdman, S. E., V. P. Rao, T. Poutahidis, M. M. Ihrig, Z. Ge, Y. Feng, M. Tomczak, A. B. Rogers, B. H. Horwitz, and J. G. Fox. 2003. CD4(⫹)CD25(⫹) regulatory lymphocytes require interleukin 10 to interrupt colon carcinogenesis in mice. Cancer Res. 63:6042–6050. 11. Eyigor, A., K. A. Dawson, B. E. Langlois, and C. L. Pickett. 1999. Cytolethal distending toxin genes in Campylobacter jejuni and Campylobacter coli isolates: detection and analysis by PCR. J. Clin. Microbiol. 37:1646–1650. 12. Eyigor, A., K. A. Dawson, B. E. Langlois, and C. L. Pickett. 1999. Detection of cytolethal distending toxin activity and cdt genes in Campylobacter spp. isolated from chicken carcasses. Appl. Environ. Microbiol. 65:1501–1505. 13. Fox, J. G. 2002. The non-H. pylori helicobacters: their expanding role in gastrointestinal and systemic diseases. Gut 50:273–283. 14. Fox, J. G., F. E. Dewhirst, Z. Shen, Y. Feng, N. S. Taylor, B. J. Paster, R. L. Ericson, C. N. Lau, P. Correa, J. C. Araya, and I. Roa. 1998. Hepatic Helicobacter species identified in bile and gallbladder tissue from Chileans with chronic cholecystitis. Gastroenterology 114:755–763. 15. Fox, J. G., F. E. Dewhirst, J. G. Tully, B. J. Paster, L. Yan, N. S. Taylor, M. J.

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