Cytolethal Distending Toxin Promotes Helicobacter cinaedi-Associated Typhlocolitis in Interleukin-10-Deficient Mice

Abstract

Helicobacter cinaedi colonizes a wide host range, including rodents, and may be an emerging zoonotic agent. Colonization parameters, pathology, serology, and inflammatory responses to wild-type H. cinaedi (WT_Hc) were evaluated in B6.129P2-IL-10^tm1Cgn (IL-10^−/−) mice for 36 weeks postinfection (WPI) and in C57BL/6 (B6) mice for 12 WPI. Because cytolethal distending toxin (CDT) may be a virulence factor, IL-10^−/− mice were also infected with the cdtB_Hc and cdtB-N_Hc isogenic mutants and evaluated for 12 WPI. Consistent with other murine enterohepatic helicobacters, WT_Hc did not cause typhlocolitis in B6 mice, but mild to severe lesions developed at the cecocolic junction in IL-10^−/− mice, despite similar colonization levels of WT_Hc in the cecum and colon of both B6 and IL-10^−/− mice. WT_Hc and cdtB mutants also colonized IL-10^−/− mice to a similar extent, but infection with either cdtB mutant resulted in attenuated typhlocolitis and hyperplasia compared to infection with WT_Hc (P < 0.03), and only WT_Hc infection caused dysplasia and intramucosal carcinoma. WT_Hc and cdtB_Hc mutant infection of IL-10^−/− mice elevated mRNA expression of tumor necrosis factor alpha, inducible nitric oxide synthase, and gamma interferon in the cecum, as well as elevated Th1-associated serum immunoglobulin G2a^b compared to infection of B6 mice (P < 0.05). Although no hepatitis was noted, liver samples were PCR positive at various time points for WT_Hc or the cdtB_Hc mutant in approximately 33% of IL-10^−/− mice and in 10 to 20% of WT_Hc-infected B6 mice. These results indicate that WT_Hc can be used to model inflammatory bowel disease in IL-10^−/− mice and that CDT contributes to the virulence of H. cinaedi.

The association of Helicobacter cinaedi infection with a variety of human diseases has received increasing attention in recent years. H. cinaedi was first isolated from homosexual men suffering from enteritis, proctitis, or proctocolitis (51). H. cinaedi was subsequently isolated from immunocompromised patients afflicted with meningitis, bacteremia, cellulitis, septic arthritis, and enteritis (2) and a neonate clinically ill with septicemia and meningitis (39), as well as from immunocompetent patients with metabolic disease (36). There are several reports of H. cinaedi isolation from various clinically healthy animal hosts, including dogs, cats, foxes, and hamsters (52). H. cinaedi has also been isolated from the inflamed colon, mesenteric lymph node, and liver of a rhesus monkey with chronic idiopathic colitis and hepatitis and from a baboon with hepatitis, as well as from feces of clinically normal, captive rhesus monkeys (11, 15, 18). Because H. cinaedi has been isolated from intestinal flora of normal hamsters and identified by PCR-based assays with wild rodents, rodents may be a zoonotic reservoir for humans (6, 47). Although H. cinaedi is the most commonly reported enterohepatic helicobacter isolated from humans, the pathogenic properties of this organism in humans or animals have not been thoroughly investigated (13).

H. cinaedi and other enterohepatic helicobacters produce a cytolethal distending toxin (CDT), which arrests cell cycle progression and causes cell death in vitro (49). CDT activity also has been associated with a variety of other gram-negative bacterial pathogens that cause human diseases affecting mucosal surfaces, such as chancroid, periodontitis, and gastroenteritis (7, 48, 56). CDT from Escherichia coli, Campylobacter jejuni, Haemophilus ducreyi, Actinobacillus actinomycetemcomitans, and enterohepatic Helicobacter species irreversibly blocks the cell cycle at the G₂/M phase of growth in a wide range of cultured cells. CDT consists of three polypeptide subunits encoded by the closely linked cdtA, cdtB, and cdtC genes. cdtB is an enzymatically active subunit which functions as a nuclease that damages DNA and triggers cell cycle arrest. cdtA and cdtC are heterodimeric subunits required for the delivery of cdtB (5, 7, 48, 55, 56). In addition to inhibiting cell cycle progression, it has been reported that C. jejuni CDT directly mediates the release of proinflammatory interleukin-8 (IL-8) from intestinal epithelial cells, suggesting that CDT has a role in the inflammatory response to mucosal infections (24).

IL-10 is an anti-inflammatory cytokine that inhibits the production of inflammatory cytokines in vitro and in vivo (38, 45). IL-10^−/− mice spontaneously develop enterocolitis when housed in conventional animal facilities but not in germfree conditions, suggesting that gut flora are critical for development of inflammation in this model (29, 46). It has been reported that Helicobacter bilis, Helicobacter hepaticus, Helicobacter typhlonius, and Helicobacter trogontum persistently colonize the lower bowel of mice and induce proliferative typhlocolitis in immune-dysregulated mice, including IL-10^−/− mice (14, 26, 30, 31, 34, 54). In this study, we used the IL-10^−/− mouse model to evaluate the pathogenic potential of H. cinaedi and the role of CDT in inducing gastrointestinal disease.

MATERIALS AND METHODS

Helicobacter cinaedi culture and DNA extraction.

The H. cinaedi wild-type strain CCUG 18818 (WT_Hc) was obtained from the Culture Collection, University of Göteborg, Sweden. WT_Hc was grown on blood agar plates (Remel, Lenexa, KS) under microaerobic conditions at 37°C for 2 or 3 days. Plates of confluent bacteria were harvested, and bacterial DNA was extracted using the High Pure PCR template preparation kit (Roche Molecular Biochemicals, Indianapolis, IN) according to the manufacturer's protocol for the isolation of nucleic acids from bacteria. Escherichia coli strain Top10 was used as a recipient for cloning, mutagenesis, and plasmid propagation and was cultured in Luria-Bertani (LB) broth or agar supplemented with ampicillin (50 μg/ml) and chloramphenicol (25 μg/ml) when appropriate (19).

Construction of two H. cinaedi isogenic cdtB gene mutants.

A 1.2-kb PCR fragment comprising the cdtB gene was amplified from H. cinaedi strain CCUG 18818 using primers HCcdt5 (5′-TAA GTG GAG AAA ATG CAG CT-3′, forward) and HCcdt3 (5′ CCA CTG CTT GTG GGA ATG-3′, reverse). The fragment was cloned into TOPO TA cloning vector pCR 2.1 (Invitrogen Carlsbad, CA). A BsmBI restriction site in the middle of the H. cinaedi cdtB gene was used to insert the chloramphenicol acetyltransferase (CAT) cassette (21). The resulting plasmid, pcdtBcm, was transformed into H. cinaedi CCUG 18818 by electroporation, and this first cdtB mutant was designated cdtB_Hc. To control for a potentially confounding spontaneous mutation during the construction of the cdtB_Hc mutant, another H. cinaedi cdtB mutant clone (cdtB-N_Hc) was constructed using the same methods by independent PCR, plasmid construction, and electroporation.

Chloramphenicol-resistant colonies were selected and further characterized by PCR. The CDT activity of WT_Hc and the cdtB mutants was assessed morphologically using Giemsa-stained HeLa S3 cells (ATCC CCL-2.2) cultured in Lab-Tek 2 chamber slides (Nalge Nunc International, Rochester, NY). Briefly, cells were incubated in 6% CO₂ at 37°C with 10 μl (1 mg/ml protein) of each filter-sterilized bacterial cell sonicate added to the chambers. Slides were incubated for 72 h, washed with phosphate-buffered saline (PBS), stained with Diff-Quick (Dade International, Miami, FL), and observed microscopically for morphological changes. The DNA content of similarly treated HeLa S3 cells was quantified by flow cytometry-based measurements of cell cycle arrest, as previously described (49). Briefly, tissue culture flasks (25 cm²) were seeded with 5 × 10⁵ HeLa S3 cells in 5 ml of Eagle minimal essential medium and 10% fetal bovine serum. After being incubated in 6% CO₂ at 37°C for 1 h, 100 μl of filter-sterilized sonicate (1 mg/ml protein) was added, and the flasks were incubated for 72 h. Cells were detached and stained with propidium iodide, and DNA analysis of 1 × 10⁴ HeLa S3 cells was performed using a FACScan flow cytometer with Cell Quant software for data acquisition and the ModFit program for data analysis (Becton Dickinson, Franklin Lakes, NJ).

Mice.

Six- to 8-week-old B6.129P2-IL-10^tm1Cgn (IL-10^−/−) mice and C57BL/6 mice (B6) (Jackson Laboratories, Bar Harbor, ME) were maintained free of known murine viruses, Salmonella spp., Citrobacter rodentium, ecto- and endoparasites, and known Helicobacter spp. in a facility accredited by the Association for Assessment and Accreditation of Laboratory Care International, under barrier conditions. Animals were housed in microisolater, solid-bottomed polycarbonate cages on hardwood bedding, fed a commercial pelleted diet, and administered water ad libitum. The protocol was approved by the Committee on Animal Care of the Massachusetts Institute of Technology.

Experimental design and infection.

In a first experiment, five IL-10^−/− mice of both genders were either sham dosed by oral gavage with broth or orally gavaged with WT_Hc or the cdtB_Hc mutant and were evaluated at 6 and 12 weeks postinfection (WPI) (Table 1). In a second experiment, five IL-10^−/− mice of each gender were similarly dosed and evaluated at 6, 12, 24, and 36 WPI. Additionally, five B6 mice of each gender were sham dosed or gavaged with WT_Hc and necropsied at 6 and 12 WPI (experiment 2). In the third experiment, five IL-10^−/− mice of each gender were shame dosed or given WT_Hc or the second cdtB isogenic mutant, cdtB-N_Hc, and were evaluated at 12 WPI for pathology and cecal colonization.

TABLE 1.

Experimental design showing numbers of mice of each genotype and infection status at necropsy time points^a

Expt no.	Mouse strain	Infection status	No. of mice at indicated WPI:
			6	12	24	36
1	IL-10−/−	Sham	5	5	ND	ND
		WTHc	5	5	ND	ND
		cdtBHc mutant	5	5	ND	ND
2	IL-10−/−	Sham	10	10	10	20
		WTHc	10	10	10	20
		cdtBHc mutant	10	10	ND	ND
	B6	Sham	10	10	ND	ND
		WTHc	10	10	ND	ND
3	IL-10−/−	Sham	ND	10	ND	ND
		WTHc	ND	10	ND	ND
		cdtB-NHc mutant	ND	10	ND	ND

^aSimilar numbers of male and female mice were used in experiment 1, and exactly equivalent numbers of males and females were used in experiments 2 and 3. ND, not done.

WT_Hc and the cdtB mutants were grown in brucella broth containing 5% fetal calf serum under microaerobic conditions, screened for morphology and motility, and resuspended in broth to approximately 10⁹ organisms/ml, as determined by spectrophotometry at A₆₆₀. Mice received 0.2 ml of fresh inoculum by gastric gavage every other day for three doses or were sham dosed with broth. Colonization with WT_Hc or the cdtB_Hc mutant was confirmed 4 WPI by PCR analysis of feces by use of previously described methods using H. cinaedi-specific primers (17). At necropsy, serum and feces samples were collected and then liver, stomach, cecum, and colon samples were collected for culture, quantitative PCR, RNA isolation, and histology.

Isolation of H. cinaedi.

Fresh feces and aseptically collected liver samples were homogenized in PBS before passage through a 0.45-μm filter onto blood agar plates for helicobacter culture. Plates were incubated at 37°C in microaerobic conditions using vented jars containing N₂, H₂, and CO₂ (80:10:10). Plates were assessed for growth after 3 to 5 days and were maintained for 2 weeks before a determination of no growth was made. Isolates were confirmed to be WT_Hc or the cdtB_Hc mutant by PCR.

Histology.

Formalin-fixed tissues were routinely processed, embedded in paraffin, cut at 4 μm, and stained with hematoxylin and eosin. Stomach and liver sections were screened for lesions but not scored, due to an absence of lesions. Large bowel lesions were scored on the basis of size and frequency of hyperplastic and inflammatory lesions on a scale of 0 to 4, with ascending severity (0, none; 1, minimal; 2, mild; 3, moderate; and 4, severe). Epithelial dysplasia and neoplasia were graded using a scale of 0 to 4: 0, normal; 1, mild dysplastic changes; 2, moderate or severe dysplasia; 3, gastrointestinal intraepithelial neoplasia; and 4, invasive carcinoma, as previously described (1, 9).

Real-time quantitative PCR for H. cinaedi colonization levels in cecum and colon.

Relative concentrations of WT_Hc and cdtB_Hc mutant DNA in the cecum and colon and cdtB-N_Hc mutant DNA in the cecum were determined by real-time quantitative PCR using the ABI Prism TaqMan 7700 sequence detection system (PE Biosystems, Foster City, CA). DNA was extracted from tissues using a High Pure PCR template preparation kit (Roche Molecular Biochemicals, Indianapolis, IN) following the manufacturer's protocol. H. cinaedi-specific primers were generated from analysis of the 16S rRNA and the 23S rRNA spacer region using Primer Express software (Applied Biosystems), with forward primer HciSPF (5′-ATG AAA ATG GAT TCT AAG ATA GAG CA-3′) and reverse primer (HciSPR 5′-AAG ATT CTT TGC TAT GCT TTT GGG GA-3′). Duplicate PCRs contained the following in 25-μl volumes: 5 μl of template DNA; 12.5 μl SYBR green master mix; 500 nm of each primer. Thermocycling was performed as follows: 50°C for 2 min and 95°C for 10 min, and then 40 repeats of 95°C for 15 s and 60°C for 60 s. Samples were also probed with 18S rRNA-based primers for quantifying host DNA (Applied Biosystems), as previously described (22, 53).

Quantitative PCR for cytokine mRNA expression profiles in cecum.

Total RNA was extracted from approximately 25 mg of mouse cecum using Trizol reagent (Invitrogen, Carlsbad, CA). RNA was further purified using the RNeasy mini kit (Qiagen, Valencia, CA) with the column-based DNase digestion protocol per the manufacturer's protocol. Total RNA (2 μg) was converted into cDNA using a high-capacity cDNA archive kit according to the manufacturer's protocol (Applied Biosystems). cDNA levels for tumor necrosis factor alpha (TNF-α), gamma interferon (IFN-γ), inducible nitric oxide synthase (iNOS), IL-6, and IL-4 mRNA were measured by quantitative PCR using commercial primers and probes for each cytokine. Briefly, duplicate 25-μl reaction mixtures contained 5 μl of cDNA, 1.25 μl of a commercial 20× primer-probe solution, 12.5 μl of 2× master mix (all Applied Biosystems), and 6.25 μl of double-distilled H₂O. Relative expression of mRNA from infected and control mice was calculated using the comparative C_T method, with RNA input standardized between samples by expression levels of the endogenous reference gene GAPDH, the glyceraldehyde-3-phosphate dehydrogenase gene. Results from duplicate samples were plotted as changes between tissues from infected and uninfected control mice.

Immunofluorescence staining for iNOS and macrophages.

Formalin-fixed cecal tissue sections were steam treated for 20 min in citrate-buffered (pH 6) target retrieval solution (Dako Cytomation, Carpinteria, CA) for epitope recovery. On an automated immunostainer (i6000, Biogenex; San Ramon CA), deparaffinized and rehydrated tissue sections were sequentially labeled for iNOS (Nos2; Santa Cruz Biotechnology, Inc., Santa Cruz, CA) and the macrophage marker F4/80 (Invitrogen, Carlsbad, CA) with streptavidin-Cy3 (Sigma, St. Louis, MO) and fluorescein (Rockland Immunochemicals, Gilbertsville, PA), respectively, as previously described (43). Slides were mounted with antifade Vectashield plus DAPI (Vector Laboratories, Burlingame, CA), and immunofluorescence was visualized with a fluorescence microscopy imaging system (Carl Zeiss Microimaging, Thornwood, NY).

ELISA for IgG2a^b and IgG1 responses to H. cinaedi.

Sera were collected at 6 and 12 WPI, and the Th1-associated immunoglobulin G2a^b (IgG2a^b) and Th2-associated IgG1 responses to antigens of H. cinaedi were measured by enzyme-linked immunosorbent assay (ELISA). Sonicated antigen from WT_Hc was coated on Immulon II plates (Thermo Labsystems, Franklin, MA) at a concentration of 10 μg/ml, and sera were diluted 1:100. Biotinylated secondary antibodies included monoclonal anti-mouse antibodies produced by clones G1-6.5 and -5.7 (PharMingen, San Diego, CA) for detecting IgG1 and IgG2a^b, respectively. Incubation with extravidin peroxidase (Sigma, St. Louis, MO) was followed by ABTS [2,2′-azino-bis(3-ethylbenzthiazoline-6-sulfonic acid)] substrate (Kirkegaard and Perry Laboratories, Gaithersburg, MD) for color development. Optical density (OD) development at 405λ was recorded by an ELISA plate reader (Dynatech MR7000, Dynatech Laboratories, Inc., Chantilly, VA).

Statistical analysis.

Data on all parameters were combined from experiments 1 and 2, and pathology was independently assessed in experiment 3. Cecal and colonic lesion scores were analyzed using the Mann-Whitney U nonparametric test for ordinal data; WT_Hc or cdtB_Hc mutant colonization, serology, and cytokine mRNA expression levels were compared by Student's t test. Values of P <0.05 were considered significant.

RESULTS

H. cinaedi cdtB isogenic mutants lost CDT activity on HeLa S3 cells.

As previously reported (49), the cell sonicate supernatant from WT_Hc applied to HeLa S3 cells induced formation of mono- and multinucleated giant cells with characteristically large nuclei (Fig. 1). In contrast, HeLa S3 cells incubated with cell sonicate supernatants from the cdtB_Hc and cdtB-N_Hc mutants maintained their normal morphology, consistent with HeLa S3 cells treated with a PBS control, indicating loss of in vitro CDT activity in H. cinaedi cdtB mutant strains. As an index of CDT-mediated cell cycle arrest, measurement of DNA content by flow cytometry indicated that WT_Hc sonicate supernatant caused 42% of HeLa S3 cells to arrest in the G₂/M phase in contrast to only 3% of HeLa S3 cells exposed to sonicate supernatant harvested from the cdtB_Hc mutant, similar to 3.4% of sham-treated cells (data not shown).

FIG. 1.

Giemsa-stained HeLa S3 cells exposed for 72 h to filter-sterilized supernatant of cell sonicates of the H. cinaedi WT strain and cdtB_Hc mutant. (Magnification, ×200.) (A) PBS control. (B) WT_Hc expressed CDT, which induced the formation of giant mono- and multinucleated cells with large nuclei. (C) The isogenic mutant (cdtB_Hc) lost CDT activity. Treated cells maintained a similar morphology to that of the PBS-treated control cells. Loss of activity was also observed using the second isogenic mutant, cdtB-N_Hc (not shown).

H. cinaedi colonization in the lower bowel was not impacted by loss of cdtB function.

All sham-dosed mice remained helicobacter-free, as confirmed by culture and PCR. WT_Hc and the cdtB_Hc mutant were isolated by culture from feces of all experimentally dosed IL-10^−/− and B6 mice at all necropsy time points, and isolates were confirmed to be WT_Hc or the cdtB_Hc mutant by PCR, including demonstration that all cdtB_Hc mutant isolates retained the CAT cassette (data not shown).

WT_Hc and the cdtB_Hc mutant colonized the cecum of IL-10^−/− and B6 mice to similar extents (Fig. 2), which was 10-fold greater than that in the colon at 6 and 12 WPI (data for colon not shown). Colonization levels of both WT_Hc and the cdtB_Hc mutant increased in the cecum between 6 and 12 WPI in IL-10^−/− mice as did WT_Hc in B6 mice (P < 0.05). In the cecum of IL-10^−/− mice, WT_Hc colonization levels peaked at 12 WPI, persisted at this level through 24 WPI, and were decreased at 36 WPI (P < 0.05), although remaining similar to the 6-WPI measurement. Male IL-10^−/− mice infected with WT_Hc had higher WT_Hc colonization levels in their cecum at 12 and 36 WPI compared to female IL-10^−/− mice (P < 0.05) (data not shown). In the third experiment, there was no significant difference in H. cinaedi cecum colonization levels between WT_Hc and the cdtB-N_Hc mutant (P = 0.37), similar to results obtained in experiments 1 and 2.

FIG. 2.

H. cinaedi colonization levels of WT_Hc or the cdtB_Hc mutant in the cecum of infected mice measured by quantitative PCR analysis at 6, 12, 24, and 36 WPI. Numbers represent the ng of H. cinaedi DNA per μg mouse DNA. Only WT_Hc and the sham control were evaluated at 24 and 36 WPI. *, P < 0.05. Bars, standard errors.

Although WT_Hc and the cdtB_Hc mutant were not recovered from liver samples by culture, approximately 33% of the liver samples from IL-10^−/− mice infected with WT_Hc were PCR positive at 6, 24, and 36 WPI, whereas only 20% of livers from B6 mice were PCR positive at 6 WPI and 10% at 12 WPI. Interestingly, livers of IL-10^−/− mice infected with the cdtB_Hc mutant were positive by PCR at 6 WPI but were negative at 12 WPI (Table 2).

TABLE 2.

Culture and PCR results from infected mice used in experiments 1 and 2

Mouse strain	Infection	Time point (WPI)	No. of samples positive/no. tested
			Fecal culturea	Liver culture	Liver PCR
IL-10−/−	WTHc	6	15/15	0/15	5/15
		12	15/15	0/15	0/15
		24	12/12	0/12	5/12
		36	17/17	0/17	5/17
	cdtBHc	6	15/15	0/15	5/15
		12	15/15	0/15	0/15
B6	WTHc	6	10/10	0/10	2/10
		12	10/10	0/10	1/10

^aAll fecal samples from infected mice were PCR positive for WT_Hc or cdtB_Hc in experiments 1 and 2 and for WT_Hc or cdtB-N_Hc in experiment 3.

WT_Hc infection induced typhlocolitis to a greater extent than the cdtB_Hc and cdtB-N_Hc mutants in IL-10^−/− mice.

No significant lesions were observed in the gastrointestinal tracts of control mice at any time point (Fig. 3A) nor in WT_Hc-infected B6 mice at 6 and 12 WPI. Stomach and liver sections from all experimental groups were screened for lesions but not scored due to absence of lesions. WT_Hc and cdtB_Hc mutant infection induced significant typhlocolitis in IL-10^−/− mice compared to uninfected controls (P < 0.001). Lesions attributable to the cdtB_Hc mutant, however, were less severe than those induced by WT_Hc infection (P < 0.05). WT_Hc infection of IL-10^−/− mice resulted in mild to severe typhlocolitis with hyperplasia, submucosal edema, epithelial defects, and dysplasia (Fig. 3C and D). Inflammation was limited to the mucosa and submucosa, consisting of segmental areas of few or moderate numbers of neutrophils, with lower numbers of histiocytes and lymphocytes. Hyperplastic crypts were lined by densely packed epithelial cells with diminished goblet cell numbers and occasional mitotic figures. There were infrequent crypt abscesses characterized by dilated crypt glands lined by attenuated epithelial cells and containing luminal cell remnants. In most IL-10^−/− mice infected with WT_Hc or the cdtB_Hc mutant, lower bowel inflammation was most severe at the cecocolic junction. Severity of typhlocolitis, accompanied by hyperplasia and dysplasia, peaked at 12 WPI (P < 0.05) and trended lower over time through 36 WPI. Infection with the cdtB_Hc mutant was not only associated with less severe inflammation at 6 and 12 WPI (P < 0.05), but only 1 of 15 cdtB mutant-infected IL-10^−/− mice developed moderate hyperplasia by 12 WPI. In contrast, WT_Hc infection resulted in significant hyperplasia and dysplasia by 6 WPI that also peaked in severity by 12 WPI, with 4 of 15 IL-10^−/− mice developing hyperplasia and dysplasia (Fig. 4B and C). Interestingly, one WT_Hc-infected IL-10^−/− mouse had developed intramucosal carcinoma of the cecum by 12 WPI (Fig. 3D and E). Lesions were less prominent in the colon of infected IL-10^−/− mice and were similar in mice infected with WT_Hc or the cdtB_Hc mutant. A limited number of focal lesions were surrounded by normal tissue (“skip lesions”) in the mid- to distal colon (Fig. 3F). Colonic lesions contained mild mucosal and submucosal inflammation, hyperplasia, edema, and occasional crypt abscesses.

FIG. 3.

Histopathology of H. cinaedi typhlocolitis in IL-10^−/− mice. (A) Cecum of sham-inoculated, disease-free control. (B) Mild or moderate inflammation in a mouse infected with the cdtB_Hc mutant. (C) Moderate typhlocolitis with mild hyperplasia, dysplasia, and crypt atrophy in a mouse infected with WT_Hc. (D and E) WT_Hc-induced gastrointestinal intraepithelial neoplasia with intramuscular dysplastic glands and deep invasion of dilated mucinous glands (arrows). (F) Focal area of epithelial hyperplasia and dysplasia bounded by normal mucosa (skip lesion) in mid-colon of a mouse infected with WT_Hc. Tissues were stained with hematoxylin and eosin. Bar = 200 μm (A to D and F) and 400 μm (E).

FIG. 4.

WT_Hc or cdtB_Hc mutant-induced pathology in IL-10^−/− mice at 6, 12, 24, and 36 WPI. The median score (horizontal bar) is indicated for each group. Only WT_Hc and the sham control were evaluated at 24 and 36 WPI. (A) Inflammation. (B) Hyperplasia. (C) Dysplasia. All infected mice had significantly higher inflammation scores than control mice (P < 0.001) at all time points. At 12 WPI, the cdtB_Hc mutant induced less inflammation than WT_Hc (*, P < 0.05).

For observing long-term effects of WT_Hc infection in IL-10^−/− mice and testing the hypothesis of whether H. cinaedi-induced typhlocolitis would progress to tumor formation in the lower bowel, WT_Hc-infected IL-10^−/− mice were monitored for up to 36 WPI (Fig. 2A, B, and C and Fig. 4). Severity of typhlitis decreased after 12 WPI (P < 0.05) and was similar at time points of 24 and 36 WPI (Fig. 4A to C). There were no significant differences in the pathology scores between female and male mice (data not shown).

In the third experiment, IL-10^−/− mice were dosed with WT_Hc or the second isogenic mutant, cdtB-N_Hc; pathology results were similar to those observed using the first isogenic mutant, cdtB_Hc. At 12 WPI, WT_Hc or cdtB-N_Hc mutant infection induced significant typhlocolitis compared to uninfected controls (P < 0.001), although lesions attributable to the cdtB-N_Hc mutant were less severe than those induced by WT_Hc infection (P < 0.05). Typhlitis, hyperplasia, and dysplasia resulted from WT_Hc infection, but dysplasia was noticeably absent in cdtB-N_Hc mutant-infected mice (Fig. 5).

FIG. 5.

Median pathology scores of the third experiment using a second isogenic mutant (cdtB-N_Hc) or WT_Hc in IL-10^−/− mice at 12 WPI. All infected mice had significantly higher inflammation scores than control mice (P < 0.001), and the cdtB-N_Hc mutant induced less pathology than WT_Hc (*, P < 0.05). Horizontal bars, median scores.

Typhlitis-associated proinflammatory cytokine and iNOS mRNA levels were increased by WT_Hc and cdtB_Hc mutant infection.

WT_Hc and cdtB_Hc mutant infection of IL-10^−/− mice stimulated increased expression of mRNA for TNF-α, iNOS, and IFN-γ above control levels at all time points (P < 0.001) (Fig. 6A to C). These proinflammatory signals peaked at 12 WPI (P < 0.05), in concert with a peak in severity of typhlocolitis (P < 0.05). In contrast, elevated TNF-α and iNOS, but not IFN-γ, mRNA expression levels in noninflamed cecal tissues from WT_Hc-infected B6 mice were similar to levels measured in samples from infected IL-10^−/− mice at 6 WPI. TNF-α and iNOS expression levels thereafter declined to control levels. There were no changes in IL-4 or IL-6 expression levels in infected IL-10^−/− or B6 mice. There were no statistically significant differences in cytokine levels between WT_Hc and cdtB_Hc mutant-infected mice.

FIG. 6.

Relative expression of select cytokine and iNOS mRNA levels in the cecum. Expression levels of TNF-α, IFN-γ, and iNOS were higher in infected IL-10^−/− mice than those in B6 mice at 12 WPI. Data represent means and standard errors (bars) of changes in mRNA expression in infected mice compared to expression in uninfected controls. *, P < 0.05; **, P < 0.01, compared with the sham control. Only WT_Hc and the sham control were evaluated at 24 and 36 WPI. There were no statistically significant differences in cytokine levels between WT_Hc- and the cdtB_Hc mutant-infected mice.

Expression of iNOS and the macrophage cell marker F4/80 were also evaluated by immunofluorescence staining in WT_Hc-infected and uninfected IL-10^−/− mice at 12 WPI. iNOS was upregulated in focal areas in the cecum of WT_Hc-infected IL-10^−/− mice compared to iNOS in control tissues; iNOS expression was highest at the base of the crypts, with increased staining also noted in the surface epithelium. Although there was a considerable infiltration of macrophages between glands of the mucosa, F4/80 staining did not colocalize with iNOS in the epithelium. The submucosa contained more scattered macrophages that were dual labeled for iNOS and F4/80 stain (Fig. 7).

FIG. 7.

Scanning laser confocal immunofluorescent detection of H. cinaedi-induced iNOS and macrophage F4/80 expression in cecal sections of WT_Hc-infected and uninfected mice. iNOS was upregulated in focal areas in the cecum of WT_Hc-infected IL-10^−/− mice. Green, F4/80; red, iNOS; blue, nucleus. (A) Control mouse. (B and C) WT_Hc-infected mice.

WT_Hc and cdtB_Hc mutant infection promoted Th1-associated IgG2a^b serological responses.

Mice infected with WT_Hc or the cdtB_Hc mutant developed significant IgG2a^b (Th1-associated) and IgG1 (Th2-associated) antibody responses by 6 WPI (P < 0.01), and the IgG2a^b response was higher in infected IL-10^−/− mice compared to infected B6 mice (P < 0.02) (Fig. 8). The IgG2a^b response predominated over the IgG1 response, especially in infected IL-10^−/− mice at 12 WPI (P < 0.05) and increased between 6 and 12 WPI (P < 0.05).

FIG. 8.

Serum antibody levels in infected IL-10 ^−/− and B6 mice against H. cinaedi whole-cell sonicate protein measured by ELISA at 6 and 12 WPI. (A) IgG1 antibody response. (B) IgG2a^b antibody response. Infection with WT_Hc or the cdtB_Hc mutant promoted significant IgG2a^b (Th1-associated) and IgG1 (Th2-associated) antibody responses by 6 WPI and increased between 6 and 12 WPI. IgG2a^b response was higher in infected IL-10^−/− mice than in infected B6 mice. *, P < 0.05. Bars, standard errors.

DISCUSSION

Inflammatory bowel disease is likely caused by genetic predisposition to pathological activation of the mucosal immune system in response to antigens derived from normal flora or infectious agents (12). Emerging evidence from animal models such as IL-10^−/− mice indicates that dysregulation of mucosal T-cell responses directed toward gut flora or specific bacteria, such as Helicobacter spp., induces harmful intestinal inflammation, with sequelae similar to pathology observed with human inflammatory bowel disease (3, 23, 30). Our results demonstrate that H. cinaedi colonized IL-10^−/− and B6 mice and caused typhlocolitis in the Th1-prediposed IL-10^−/− strain, similar to other models using IL-10^−/− mice infected with enterohepatic helicobacters (14, 32, 33). Also consistent with other Helicobacter species infections in IL-10^−/− mice, the H. cinaedi-induced inflammation, hyperplasia, and dysplasia were most severe at the cecocolic junction, where colonization of H. cinaedi was greatest. The development of typhlitis was associated with concurrent proinflammatory cytokine and iNOS responses and a predominant IgG2a^b serologic response to H. cinaedi infection, which indirectly reflects a proinflammatory response of mice on a B6 background (35). Additionally, IL-10^−/− mice infected with the independently generated cdtB_Hc and cdtB-N_Hc isogenic mutants developed less-severe disease, providing support that CDT is a virulence factor of H. cinaedi.

Diarrhea has been associated with H. cinaedi infection in a dog and nonhuman primates as well as hepatitis in a baboon and rhesus monkey (15, 18, 37). Hepatitis was not observed nor expected in this study because of the known resistance of B6 mice to helicobacter-associated hepatitis (25). Positive H. cinaedi PCR results for 10 to 33% of the liver samples suggest either that WT_Hc and the cdtB_Hc mutant translocated to the liver or that, at a minimum, their DNA was present in the liver, potentially the result of enterohepatic circulation from inflamed intestines with increased permeability. H. cinaedi infection should be evaluated with other mouse strains, such as the A/JCr strain that develops mild typhlitis and, more importantly, chronic active hepatitis and hepatocellular carcinoma in response to H. hepaticus infection (17). Although we have no evidence of sustained extraintestinal disease in the mice evaluated in this study, H. cinaedi is increasingly associated with extraintestinal infections in humans, often in patients compromised by surgery (28) or other serious metabolic and immunocompromising infectious diseases (11, 13, 15, 36, 52).

This is the first description of H. cinaedi-induced typhlocolitis in a mouse model. Notably, H. cinaedi infection has been implicated by PCR in some wild South American rodents (6) but has not been isolated by culture from naturally infected mice used in research (50). Like H. pylori, H. cinaedi was able to persistently colonize IL-10^−/− mice for at least 36 weeks, despite the host inflammatory response (8). In the absence of IL-10-mediated immunoregulation, H. cinaedi-induced disease was characterized primarily as chronic typhlitis with secondary hyperplasia and dysplasia, similar to lesions induced by H. hepaticus in IL-10^−/− mice (57). The colon was not uniformly inflamed; rather, a pattern of “skip lesions” developed with inflamed segments surrounded by noninvolved colon; these lesions resemble the segmental pathology characteristic of Crohn's disease (44).

The H. cinaedi isogenic mutants cdtB_Hc and cdtB-N_Hc colonized IL-10^−/− mice to the same extent as wild-type H. cinaedi (WT_Hc) but induced less-severe lesions. Several other Helicobacter species, including H. hepaticus, H. bilis, and H. pullorum, also express CDT (4, 49, 56). Other enteric bacteria-producing CDT include E. coli, Shigella spp., and Campylobacter spp., which are also associated with enteritis. The CDT mutants of both H. hepaticus and C. jejuni have been used with mouse models to investigate the function of CDT, and the results have varied, based on the mouse strain. Ge et al. demonstrated that a H. hepaticus CDT mutant lost colonization in female Swiss Webster and A/JCr mice much faster than in male mice (19, 20). In another study, C. jejuni cdtB mutant strain 81-176 was less efficient than wild-type C. jejuni in colonizing C57BL/129 mice and was cleared by the host within 4 months (16). In contrast, our results indicated persistent, similar intestinal colonization levels of both WT_Hc and the cdtB_Hc mutant in both genders of IL-10^−/− mice. However, it appears that the cdtB_Hc mutant, unlike WT_Hc, did not persist in the liver (as judged by PCR) in IL-10^−/− B6 mice.

More importantly, isogenic mutants of other bacterial pathogens that normally express CDT have been shown to lose virulence in vivo. When orally administered to C.B.-17-SCID-Beige mice, the cdtB mutant and wild-type C. jejuni both produced similar colonization levels; however, the cdtB mutant was impaired in its ability to cause bacteremia and invade the spleen and liver, suggesting that CDT may be important for tissue invasion (42). In NF-κB-deficient (3×) mice (on a 129 × C57BL/6 background), the C. jejuni cdtB mutant induced significantly less gastritis than wild-type C. jejuni (16). These results are comparable to the attenuated lower bowel lesions observed with IL-10^−/− mice infected with a H. hepaticus CDT mutant (41). In our study, colonization was not impacted by CDT, but IL-10^−/− mice infected with the cdtB_Hc or cdtB-N_Hc mutant developed significantly less severe typhlocolitis compared to mice infected with WT_Hc. These results indicate that CDT expression may not always be necessary for colonization of mice, but loss of function has been associated with amelioration of disease, as reported for C. jejuni, H. hepaticus, and now, H. cinaedi.

Our data on cytokine expression levels in inflamed tissues are consistent with other reports that IL-10^−/− mice infected with helicobacters develop Th1-mediated typhlocolitis associated with CD4⁺ T-cell production of IFN-γ, TNF-α, and iNOS expression (30, 32, 40). Upregulated iNOS mRNA is associated with significant levels of nitric oxide (NO), which promotes inflammation and tissue injury (10, 27). Only IL-10^−/− mice developed typhlocolitis from H. cinaedi infection, while WT_Hc-infected B6 mice did not develop disease. In B6 mice, regulatory T-cell inhibition of colitis was most likely mediated, at least in part, by secretion of IL-10. Levels of colonization, proinflammatory cytokine expression, and severity of pathology were all correlated with progression of disease in the IL-10^−/− mice through 12 WPI, after which these parameters regressed in magnitude.

In summary, we observed that H. cinaedi colonized B6 and IL-10^−/− mice on the same background but induced disease only in IL-10^−/− mice, consistent with other reports using enterohepatic Helicobacter species infections in IL-10^−/− mice. Because H. cinaedi cdtB mutants caused less disease, the availability of this model will allow further in vivo investigations of H. cinaedi pathogenesis and particularly of CDT as a virulence factor promoting disease.