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Comp Med. Dec 2008; 58(6): 534–541.
Published online Dec 2008.
PMCID: PMC2710754

Helicobacter typhlonius and Helicobacter rodentium Differentially Affect the Severity of Colon Inflammation and Inflammation-Associated Neoplasia in IL10-Deficient Mice

Abstract

Infection with Helicobacter species is endemic in many animal facilities and may alter the penetrance of inflammatory bowel disease (IBD) phenotypes. However, little is known about the relative pathogenicity of H. typhlonius, H. rodentium, and combined infection in IBD models. We infected adult and neonatal IL10−/− mice with H. typhlonius, H. rodentium, or both bacteria. The severity of IBD and incidence of inflammation-associated colonic neoplasia were assessed in the presence and absence of antiHelicobacter therapy. Infected IL10−/− mice developed IBD with severity of noninfected (minimal to no inflammation) < H. rodentium < H. typhlonius < mixed H. rodentium + H. typhlonius (severe inflammation). Inflammation-associated colonic neoplasia was common in infected mice and its incidence correlated with IBD severity. Combined treatment with amoxicillin, clarithromycin, metronidazole, and omeprazole eradicated Helicobacter in infected mice and ameliorated established IBD in both infected and noninfected mice. Infection of IL10−/− mice with H. rodentium, H. typhlonius, or both organisms can trigger development of severe IBD that eventually leads to colonic neoplasia. The high incidence and multiplicity of neoplastic lesions in infected mice make this model well-suited for future research related to the development and chemoprevention of inflammation-associated colon cancer. The similar antiinflammatory effect of antibiotic therapy in Helicobacter-infected and -noninfected IL10−/− mice with colitis indicates that unidentified microbiota in addition to Helicobacter drive the inflammatory process in this model. This finding suggests a complex role for both Helicobacter and other intestinal microbiota in the onset and perpetuation of IBD in these susceptible hosts.

Abbreviations: IBD, Inflammatory bowel disease

Inflammatory bowel disease (IBD) is hypothesized to develop due to aberrant immune responses induced by gut microbes.5 IBD does not occur in germ-free IL10−/− mice,2,15 indicating the importance of microorganisms as environmental triggers of intestinal inflammation. However, conventionally colonized or specific pathogen-free IL10−/− mice may develop colitis spontaneously2,32 or in response to specific triggers such as nonsteroidal antiinflammatory drugs3,14 or infections with certain bacteria.6,16,18 The normal lack of ongoing immune responses against bacteria in subjects without IBD has been attributed to the immunologic tolerance that specifically downregulates immune responses against antigens derived from these bacteria. Nevertheless, despite a large number of studies, no single bacterial type has fulfilled Koch postulates and been confirmed as a cause of IBD in animals or humans.

Previous studies used fluorescence in situ hybridization with probes specific for bacterial 16S rRNA combined with conventional histologic techniques to study the relationships between various species of intestinal bacteria and the mucosa in mice and humans with IBD.33,34 Those studies showed that in normal mice, most bacterial groups are separated from the mucosal surface by either a mucus layer that excludes bacteria or, in the cecum and proximal colon, by an ‘interlaced’ layer that is composed of tightly packed bacteria. The interlaced or mucus layer thus limits the contact of the bulk of the enteric bacteria with the mucosal epithelium. In contrast, complex biofilms composed of multiple species of bacteria that were firmly adherent to the mucosal surface were identified in the majority of colon tissue samples collected from humans and mice with IBD.33,34 The presence of a biofilm abrogates the protective effects of the normal layer of mucus and can allow luminal bacterial antigens and toxins to reach the unshielded epithelial surface, where they can trigger cascades of host inflammatory responses. Situations that cause defects in the epithelial surface or degrade the protective qualities of mucus or the interlaced layer (or both) may allow contact of bacterial antigens and adjuvants with immune cells located in the lamina propria and lead to the generation of immune responses that result in IBD.34

Helicobacter species are used frequently to model microbial triggers of colon inflammation, because they have previously been linked to the development of both IBD- and inflammation-associated neoplasia.11,21,29 Most studies have been performed by using Helicobacter hepaticus or H. bilis.20 However, H. typhlonius, H. rodentium, H. muridarum, H. ganmani, H. trogontum and other species8,12,17,29,35 can also be endemic in research animal facilities. The pathophysiologic effects of these less-common Helicobacter species are, for the most part, poorly investigated.

Most rodent Helicobacter species are urease-negative and therefore preferentially colonize the intestine, but some species produce urease enzyme and can translocate to the liver or colonize the biliary system.13 H. typhlonius was shown to cause an enteric disease characterized by mucosal hyperplasia and associated inflammation in the cecum and colon in immunodeficient mice11,23 and IL10−/− mice.18 H. typhlonius is genetically related most closely to H. hepaticus, having only 2.36% difference in the 16S rRNA gene sequence, but H. typhlonius has a unique intervening sequence in this gene that makes it easily recognizable by PCR.9,12 Molecular detection of this pathogen with PCR is rapid, sensitive and allows the detection of the early phases of infection; further enhanced sensitivity is achieved with nested primers.22 One of the most important features of PCR is that it can be performed noninvasively on fecal pellets. Data regarding the pathogenetic mechanisms of H. rodentium are scarce.35,36 H. rodentium alone apparently does not cause hepatitis or enteritis in A/JCr or C.B-17/IcrCrl-scidBr mice; however, coinfection with H. hepaticus and H. rodentium was associated with augmented cecal gene expression and clinical diarrheal disease in immunodeficient mice compared with mice infected with H. hepaticus alone.23

Previous reports demonstrated that H. typhlonius was capable of initiating colitis in adult IL10−/− mice.10,11 In those studies, colitis was relatively mild, with no development of inflammation-associated neoplasia. H. rodentium has been described to be nonpathogenic in adult wild-type mice but did enhance cytokine production in the cecum of mice also infected with H. hepaticus.23 We recently observed rapid onset of severe IBD and a high incidence of inflammation-associated neoplasia in IL10−/− mice that were coinfected with both H. typhlonius and H. rodentium as pups.16 The current study was undertaken to determine the relative roles of H. rodentium and H. typhlonius, individually and in combination, and age at infection in the development of colon inflammation and inflammation-associated neoplasia in IL10−/− mice. Novel features of our model include controlled infection of the combination of H. typhlonius and H. rodentium9 and infection of IL10−/− mice during the neonatal period.

Materials and Methods

Animals and husbandry.

Specific pathogen-free IL10−/− male and female mice on the C57BL/6 background (strain name = B6.129P2-Il10tm1Cgn/J; stock # 002251) were obtained from Jackson Laboratories (Bar Harbor, ME). Mice were confirmed to be Helicobacter-free by PCR using a genus-specific primer. Mice were housed in polycarbonate microisolation caging in ventilated isolation units or on individually ventilated racks under BSL2 conditions, with access to food and water ad libitum. Mice were observed daily for clinical signs of distress, and weight was monitored 3 times per week. Humane endpoints included loss of more than 15% of body weight or development of rectal prolapse, a well-recognized complication of chronic inflammation in the colon. The success of this study was dependent on stringent husbandry techniques to prevent cross-contamination. These techniques included a strictly enforced order of cage handling and scrupulous attention to environmental sanitization. All mice were consistently negative for all except the intentionally introduced Helicobacter spp. All animal studies were approved by the Duke University Institutional Animal Care and Use Committee.

Helicobacter infection.

Mice were infected on day 0 with H. typhlonius (clinical isolate DU-01),16 H. rodentium (MIT 95-1707; equal to ATCC type strain 700285)29 by gavage of a single dose of 500 µl culture (approximately 5 × 107 organisms unless otherwise specified). For inoculation, both strains were grown in Brucella broth (Becton Dickinson, Franklin Lakes, NJ) as previously described.19 Cultures were agitated with a stir bar in a 250-ml Erlenmeyer flask and were incubated for 24 h at 37 °C in a an atmosphere of 90% N2, 5% H2, and 5% CO2. Noninfected controls were sham-gavaged or received sterile broth or PBS only. Because initial experiments showed frequent failure of sustained H. rodentium infection when H. rodentium and H. typhlonius were given simultaneously, an additional dose of H. rodentium was given on day 2 to mice in the mixed-infection group. Infection was confirmed at 1 wk after infection and at 4-wk intervals thereafter by analysis of feces by PCR (see PCR of Helicobacter organisms). Breeding to generate neonatally infected mice typically began immediately after infection and was performed in triads consisting of 1 male and 2 female mice. Infection in pups born to infected dams was confirmed 1 to 2 wk postweaning. Mice were euthanized by CO2 asphyxiation1 if they developed 15% body weight loss or rectal prolapse or when they reached 7 to 8 months of age. All mice in this study were evaluated pathologically for both colitis and neoplasia. Sentinel mice exposed repeatedly to dirty bedding from the mice used in this study were negative for parasites by microscopic exam, negative for Citrobacter rodentium by fecal culture, and negative by serology for a panel of 22 murine protozoal, bacterial, and viral pathogens, including murine parvovirus, murine hepatitis virus, and murine norovirus. The studies were performed at an AAALAC-accredited institution. All studies were performed in accordance with The Guide for the Care and Use of Laboratory Animals.24

AntiHelicobacter therapy.

Mice designated to receive an antiHelicobacter therapy treatment received commercially available wafers containing 3 mg amoxicillin, 0.5 mg clarithromycin, 1 mg metronidazole, and 20 µg omeprazole per 5 g of food (BioServ, Frenchtown, NJ). Mice were maintained on antiHelicobacter wafers until euthanasia for tissue harvest.

To analyze the effects of antiHelicobacter therapy on noninfected IL10−/− mice with colitis, the mice were given 200 ppm piroxicam for 7 d to trigger development of chronic colitis and then were treated with the 4-drug antiHelicobacter therapy combination for 16 d.

Sample collection.

After euthanasia, the digestive tract from stomach to anus was removed and divided into segments representing the stomach, proximal, mid-, and distal small intestine, cecum, and proximal, mid-, distal, and terminal colon and rectum. Other organs collected to assess the extent of infection were mesenteric lymph nodes, spleen, liver, pancreas, stomach, ovary, and lung. Portions of each gastrointestinal segment were rinsed briefly with PBS to remove nonadherent organisms. Tissues for molecular analysis were frozen immediately and stored at −20 °C for subsequent quantitation of associated Helicobacter organisms by quantitative real-time PCR. The remaining tissues were fixed in Carnoy solution for 2 to 4 h and then processed and embedded into paraffin.

PCR detection of Helicobacter organisms.

DNA was extracted from weighed fecal and tissue samples by using the DNeasy Tissue Kit (Qiagen, Valencia, CA) according to the manufacturer's instructions. Briefly, samples were thawed in 180 µl ATL buffer and 20 µl proteinase K were added and then incubated overnight at 56 °C. Next, 200 µl AL buffer was added to the tube, the sample was vortexed, and 200 µl 100% ethanol was added. Finally, sample was loaded onto the silica-gel column, washed, and eluted in 100 µl elution buffer. To quantify the relative concentrations of fecal and mucosa-associated H. rodentium and H. typhlonius organisms, DNA samples were probed with primers designed to amplify segments of the H. rodentium Ni/Fe hydrogenase or H. typhlonius cdtB genes.28 A standard curve for quantification of the 2 Helicobacter strains was generated from serial dilutions of bacterial DNA and used to calculate numbers of bacterial copies per gram of tissue or feces.

Estimates of the number of Helicobacter genome copies in the standard were based on a genome size of 1.8 megabases and a molecular mass of 1.09 × 109 Da. The PCR reactions and melting curves were performed in 20 µl that contained 0.5 µl of each primer, 10 µl SYBR Green PCR Master Mix (Stratagene, La Jolla, CA), and 4 µl sample DNA. The PCR reaction was incubated at 95 °C for 15 min to activate the polymerase followed by 40 cycles consisting of denaturation for 15 s at 94 °C, annealing for 20 s 58 °C, and extension for 30 s at 72 °C. Fluorescence was monitored at the end of each extension phase. After amplification, melting curves were generated to verify PCR product identity.

Histologic scoring.

The severity of colonic inflammation and incidence of colon neoplasia seen in hematoxylin and eosin-stained sections was scored by a pathologist blinded to treatment group. Histologic scores were calculated (as described in reference 15 and modified from reference 6) by using a scale that takes into account mucosal changes in 5 different bowel segments, including hyperplasia and ulceration, degree of inflammation, and percentage of each bowel segment affected by these changes. With this scale, the maximum score is 75, and a score greater than 12 indicate the presence of colitis. Sections also were scored for neoplasia according to a consensus report and recommendations.4 Gastrointestinal intraepithelial neoplasia (synonymous with atypical hyperplasia, microadenoma, and carcinoma in situ) and adenoma were considered to be noninvasive lesions. Invasive lesions were classified as adenocarcinoma.

Statistical analysis.

Statistical comparison of groups was performed by using Student t tests or ANOVA. Survival rates were calculated by using the Kaplan–Meier test with P values calculated by using the log-rank test. A P value of less than or equal to 0.05 was considered to be significant.

Results

Infection with H. typhlonius with or without H. rodentium markedly increases the severity of IBD in adult IL10−/− mice.

IL10−/− mice at 6 to 8 wk of age were infected with either H. typhlonius, H. rodentium, or both and then observed for as long as 82 d before histologic determination of IBD severity. Histologic scores showed marked differences in colonic inflammation between various infection groups (Figure 1 B). Control IL10−/− mice did not develop inflammation during this time period (mean histologic score ± SEM, 10 ± 3; n = 9). In contrast, mice infected with H. rodentium or H. typhlonius or both developed colonic inflammation. Mice infected with H. rodentium alone developed mild to moderate colitis (21 ± 5; n = 10). Mice infected only with H. typhlonius had histologic scores of 29 ± 3 (n = 9; P = 0.0005 versus noninfected controls), whereas those infected with both H. rodentium and H. typhlonius had histologic scores of 38 ± 5 (n = 7; P = 0.0009 versus noninfected controls). Inflammatory changes were noted in all portions of the colon in these mice but were most severe in the cecum, followed by the proximal colon and terminal colon and rectum.

Figure 1.
Short-term survival and colitis severity in Helicobacter-infected adult IL10−/− mice. (A) Kaplan–Meier plot shows survival rate of adult IL10−/− mice infected with H. rodentium (H. rod), H. typhlonius (H. typh), ...

Some infected mice developed rectal prolapse during the course of this study and required euthanasia for humane reasons. The mean survival rate for the mice infected with H. typhlonius was significantly different than that of noninfected controls (P = 0.017; Figure 1 A), whereas the mean survival of mice infected with H. rodentium was not statistically different (P = 0.3) from that of noninfected controls. Overall, the severity of colitis in these Helicobacter-infected mice was higher than that seen in other murine models of IBD, including piroxicam-triggered colitis in IL10−/− mice.14 Although the mean histologic score in mice infected with H. rodentium was not statistically different from that seen in control mice in light of the wide variability of inflammation severity in the H. rodentium group, some mice in that group had severe IBD. Therefore, H. rodentium is capable of triggering IBD in IL10−/− mice. Colonic neoplastic lesions were not detected during this short-term experiment.

To further address the contribution of these specific Helicobacter species to colitis severity and inflammation-associated colonic neoplasia, additional cohorts of adult IL10−/− mice were infected with H. rodentium or H. typhlonius and monitored for as long as 7 mo postinfection. All control noninfected mice survived to 28 wk of age and demonstrated minimal to no intestinal inflammation at this time point (mean histologic score ± SEM, 11 ± 1; n = 10; Figure 2 B). In contrast, 50% of mice infected with H. rodentium and 100% of mice infected with H. typhlonius developed rectal prolapse that required euthanasia for humane reasons prior to the scheduled end point at 28 wk of age (Figure 2A). Mean survival for mice infected with H. rodentium was 17 ± 3 wk (P = 0.01 versus noninfected), with corresponding mean survival of 10 ± 2 wk for mice infected with H. typhlonius (P = 0.0001 versus noninfected).

Figure 2.
Long-term survival and colitis severity in Helicobacter-infected adult IL10−/− mice. (A) Kaplan–Meier plot shows survival rate of adult IL10−/− mice (n = 10 for each group) infected with H. rodentium (H. rod) or ...

The incidence of colonic neoplasia in mice infected with H. typhlonius was 50% whereas 40% of the total mice studied had invasive adenocarcinoma (Figure 3 K and L; Figure 4). The incidence of neoplasia tended to increase with time after infection. Therefore the high incidence of rectal prolapse that necessitated early euthanasia likely decreased the frequency of neoplasia. In addition, the incidence of colonic neoplasia decreased as the severity of IBD decreased, with 30% incidence of gastrointestinal intraepithelial neoplasia in mice infected with H. rodentium alone. None of these H. rodentium-infected mice developed invasive adenocarcinoma.

Figure 3.
Colon histology and neoplasia in Helicobacter-infected and noninfected IL10−/− adult mice. The histologic appearance of the cecum is shown for noninfected control mice (A, B), mice infected with H. rodentium (H. rod; C, D), H. typhlonius ...
Figure 4.
Incidence of neoplasia in IL10−/− mice born to mothers infected with H. rodentium (H. rod), H. typhlonius (H. typh), or both (Mixed). Gray bars represent mice with only noninvasive gastrointestinal intraepithelial neoplasia (equivalent ...

Effects of infection of pups with H. typhlonius, H. rodentium, or both species on IBD severity in IL10−/− mice.

A previously published study showed an extremely high incidence (95%) of colonic neoplasia at a mean age of 21 ± 2 wk in IL10−/− mice infected with a combination of H. typhlonius and H. rodentium via exposure to their infected dams.16 In the current set of experiments, female mice were infected with H. rodentium, H. typhlonius, or both prior to initiation of pregnancy, and pups were naturally infected by exposure to maternally excreted organisms. Interestingly, mice infected as pups had a decreased incidence of rectal prolapse that necessitated euthanasia compared with mice infected with the same Helicobacter species as adults (P = 0.007 for H. rodentium and H. typhlonius infections, compare Figures 2 A and 5 A). Mice infected with these Helicobacter species as pups and monitored for as long as 7 mo showed a similar pattern of survival and histologic scores compared with adults. Mice infected with H. typhlonius as pups showed significantly lower length of survival (19 ± 1 wk; n = 19) than that of controls (P < 0.01; Figure 5A) and higher inflammation scores (mean ± SEM, 42 ± 3; n = 17; Figure 5 B) than did the other groups. The mean length of survival for mice infected with H. rodentium as pups was 29 ± 1 wk of age (n = 18; P = 0.05 compared with noninfected), allowing development of inflammation-associated neoplasia. In mice infected with H. typhlonius as pups, neoplastic lesions were present in 24% of mice examined, with invasive adenocarcinoma present in 18% (Figure 4). Neoplasia was present in 29% of mice infected with H. rodentium as pups, with 12% having invasive adenocarcinoma.

Figure 5.
Survival and colitis severity in IL10−/− mice infected as pups. (A) Kaplan–Meier plot showing survival rate of pups born to the mothers infected with H. rodentium (H. rod), H. typhlonius (H. typh), or both (Mixed). Control, H. ...

Antibiotics eradicated Helicobacter infection and decreased se­verity of IBD in Helicobacter-infected mice.

To address the role of Helicobacter organisms themselves in driving intestinal inflammation, we treated IL10−/− mice infected with H. typhlonius, H. rodentium, or both as adults with antiHelicobacter therapy beginning on day 30 after infection. Mice were euthanized for histologic determination of colon inflammation severity after either 7 or 10 wk of treatment. Fecal PCR showed eradication of detectable fecal excretion of Helicobacter DNA by day 7 of antiHelicobacter therapy, with fecal PCR remaining negative throughout the remainder of the study. At the time of euthanasia, mice infected with Helicobacter but not treated with antibiotics had moderate to severe colitis (Figure 6). After 7 to 10 wk of combination antiHelicobacter therapy, the mice previously infected with Helicobacter spp. had mild or no colitis, with histologic scores that were statistically similar to antibiotic-treated but noninfected control IL10−/− mice (Figure 6). These histologic scores did not differ from those of noninfected IL10−/− mice without colitis. Euthanasia due to rectal prolapse typically would be necessary for a number of mice infected with these Helicobacter species but not treated with antibiotics (compare Figures 1 A and 5 A). However, no rectal prolapse or other cause of mortality occurred in infected, antibiotic-treated mice in this experiment. Therefore, treatment with antiHelicobacter therapy decreased the severity of colitis in the Helicobacter-infected mice and increased the survival rate in all infected experimental groups.

Figure 6.
Effect of antiHelicobacter therapy on Helicobacter-infected IL10−/− mice. Histologic scores (mean ± SEM) are shown for IL10−/− mice infected with H. rodentium or H.typhlonius at the age of 6 to 8 wk and treated ...

AntiHelicobacter therapy decreased IBD severity in noninfected IL10−/− mice.

The combination of antibiotics used to treat mice in the previous experiment was designed to eradicate Helicobacter spp. but likely also has effects on other nonHelicobacter intestinal microbiota that may affect the severity of intestinal inflammation. To address this question, IL10−/− mice confirmed by PCR to be noninfected with Helicobacter but with similarly severe IBD triggered by a 7-d exposure to 200 ppm piroxicam in food3,14 were treated for 16 d with the same 4 drug antiHelicobacter therapy combination used in the Helicobacter-infected mice. Helicobacter-noninfected IL10−/− mice with IBD triggered by piroxicam exposure showed minimal to no colitis after treatment with the antiHelicobacter therapy combination for 16 d (mean histologic score ± SEM, 11 ± 3; n = 10; Figure 7). In contrast, noninfected IL10−/− mice with IBD triggered by piroxicam that were not treated with antiHelicobacter drugs had moderate to severe colitis at this time point (mean score ± SEM, 31 ± 5; n = 8; P = 0.006).

Figure 7.
Effect of antiHelicobacter therapy on noninfected IL10−/− mice with colitis. Inflammatory bowel disease was induced by exposure to 200 ppm piroxicam for 7 d, after which mice were treated with the 4-drug antiHelicobacter therapy combination ...

Helicobacter infection status of organs in infected mice.

An abundance of data is available regarding infection with H. bilis and H. hepaticus in the colon of IL10−/− mice,10,25 but little is known about other Helicobacter species including H. typhlonius and H. rodentium. In addition, most previous studies relied primarily on qualitative analysis of Helicobacter presence in feces. However, the anatomic location of infection and the bacterial burden may affect the severity of inflammation and can only be evaluated by direct analysis of tissue. Therefore we analyzed freshly passed feces, contents of the gastrointestinal tract, and a broad range of tissues harvested from Helicobacter-infected and control noninfected mice on day 82 postinfection for Helicobacter DNA by quantitative real-time PCR (n = 3 mice/group). As expected, both H. rodentium and H. typhlonius were detected in feces from infected mice (Figure 8). More importantly, these organisms frequently were detected in washed tissue samples from the stomach and all portions of the colon. H. rodentium DNA also was detected in multiple samples from the small intestine and in 1 mesenteric lymph node sample. Analysis of the contents of various anatomic regions of the gastrointestinal tract mimicked the distribution of the Helicobacter in the tissue (data not shown), with more bacteria in the contents compared with the tissue. Detection of Helicobacter DNA in washed tissue samples suggests that some Helicobacter organisms adhere strongly to the mucosal surface, whereas detection in feces indicates that others readily pass into the fecal stream.

Figure 8.
Quantification of H. rodentium and H. typhlonius in intestinal tissue and stomach. Three IL10−/− mice inoculated with both bacterial strains were studied. Tissue was collected on day 82 of infection. Quantification of the bacterial genomes ...

Discussion

Our results showed increased colon inflammation in IL10−/− mice infected with H. typhlonius with or without H. rodentium, compared with noninfected mice. Infection with H. rodentium alone was also capable of triggering IBD in IL10−/− mice. Compared with that in noninfected mice, survival was decreased in adults infected with H. typhlonius, either alone or together with H. rodentium. Mice infected with these Helicobacter species as pups had qualitatively similar severity of inflammation compared with those infected as adults, however long-term survival was much

Our studies show that the clinical severity of disease is affected by whether Helicobacter infection is acquired before (as pups) or after establishing normal endogenous microbiota. In our study, mice infected as pups developed inflammation which resembled that of adults inoculated with the live cultures, but the survival rate of mice infected as pups was much higher and the incidence of inflammation-associated neoplasia was decreased relative to outcomes in the adult study. More studies are necessary to determine the explanations for these observations. Direct infection of adult mice with known concentrations of bacteria may change the colonic microenvironment in ways that affected the development of inflammation in that study group. It is possible that mice infected as pups could develop partial resistance to Helicobacter pathogenicity before being exposed to normal environmental microbiota; if so, it seems that such resistance did not eliminate the risk of IBD.

This experiment shows that H. typhlonius and H. rodentium can trigger the development of IBD in IL10−/− mice, although the mechanism remains somewhat unclear. Because bacterial antigens are thought to drive IBD, H. rodentium and H. typhlonius may burrow through the mucus to grow adjacent to the intestinal epithelial surface, where they degrade the barrier properties, similar to the documented action of H. pylori in the stomach.29 The injurious leakage in this case would be of bacterial antigens and adjuvants that incite immune response that damage the intestine rather than acid (as in mechanism of H. pylori), thereby leading to the development of IBD in susceptible host. That antiHelicobacter treatment also completely abrogated established intestinal inflammation in the noninfected, piroxicam-treated IL10−/− mice suggests that this antiHelicobacter treatment also acts on additional (nonHelicobacter) bacteria that drive the disease in noninfected mice.

Overall, mice directly infected with H. typhlonius as adults had a higher incidence of neoplasia compared with the mice infected as pups through contact with the infected mother (Figure 4), whereas mice infected as adults with H. rodentium did not develop invasive carcinoma at all. Interestingly, our previous studies16 showed a very high incidence of inflammation-associated colonic neoplasia in IL10−/− mice coinfected with H. typhlonius and H. rodentium as pups (mean of 4 neoplastic lesions per colon in 14 mice examined, with invasive adenocarcinoma present in 57%). The lower number of neoplastic lesions in mice infected with H. rodentium as adults in the present study may suggest lower pathogenicity of H. rodentium as reported previously.23 In addition, we show a high rate of neoplasia in mice infected with H. typhlonius alone as pups, but the incidence and multiplicity of neoplastic lesions was less than reported previously for mixed infections with both H. typhlonius and H. rodentium.16 The combination of H. rodentium and H. typhlonius may be critical for the early onset and severity of inflammation observed in previous studies.16

Helicobacter species have been shown to infect organs of the gastrointestinal tract, including stomach, intestine, and liver.26,31 The murine Helicobacter species H. typhlonius and H. rodentium have generally been considered to colonize the cecum. However, the PCR studies reported here demonstrate that the range of infected sites is much broader than previously thought.16,33 In the present study, we detected both H. rodentium and H. typhlonius (especially in the mice initially infected with both strains) in the tissue collected from several organs, including stomach. The detection of Helicobacter DNA in gastric contents may simply reflect coprophagia. However, detection of Helicobacter DNA in washed gastric tissue suggests that the organisms are tightly associated with the mucosa. Furthermore, the levels of Helicobacter organisms detected in gastric tissue (5.5 × 107 and 1.7 × 106 organisms per g of tissue for H. rodentium and H. typhlonius, respectively) seem rather higher than might be expected to be due to coprophagia alone. Although H. rodentium and H. typhlonius are urease-negative, they have been previously been reported to be associated with gastric mucosa.7,27 Other investigators27 detected H. typhlonius in the feces, gastric, and intestinal tissues and reproductive organs of wild-type C57BL/6 mice at wk 8 to 10 postinfection. However, the mice in that study were infected naturally, and the inoculum size is unknown. H. rodentium and H. typhlonius may synergize, thus allowing competitive exclusion necessary for survival in variety of tissues. Note that the PCR assays used in our studies detect bacterial DNA and cannot address the viability of the bacteria detected.

In conclusion, infection with H. rodentium, H. typhlonius, or both species markedly increased the development and severity of colon inflammation and inflammation-associated colonic neoplasia in IL10−/− mice. The rapid development of IBD makes this model very suitable for the studies on the mechanisms of IBD pathogenesis and treatment. In addition, mice coinfected with either or both organisms as pups may be useful for studies of chemoprevention of IBD-associated colonic neoplasia. IL10-deficient mice produce all lineages of immune cells and mount very strong responses to immune challenges because of their lack of the immunoregulatory cytokine IL10. The demonstration that H. rodentium can be pathogenic in these mice that are not classically immunodeficient clearly indicates that lab animal veterinarians should consider this organism to be a pathogen rather than a commensal organism. Because the reliability of an experiment that uses an in vivo model system depends on understanding and controlling all variables that can influence the experimental outcome, unintentional or nonrecognized infections with H. rodentium or H. typhlonius might interfere with ongoing research studies.

Acknowledgments

The authors would like to acknowledge the expert technical assistance of Chau T Trinh and Paula K Greer. This work was supported by National Institutes of Health grant R01-CA115480 to LPH.

References

1. American Veterinary Medical Association 2007. AVMA guidelines on euthanasia (formerly Report of the AVMA Panel on Euthanasia): Jun 2007 update. Available from http://www.avma.org/issues/animal_welfare/euthanasia.pdf
2. Berg DJ, Davidson N, Kuhn R, Muller W, Menon S, Holland G, Thompson-Snipes L, Leach MW, Rennick D. 1996. Enterocolitis and colon cancer in interleukin-10-deficient mice are associated with aberrant cytokine production and CD4+ TH1-like responses. J Clin Invest 98:1010–1020 [PMC free article] [PubMed]
3. Berg DJ, Zhang J, Weinstock JV, Ismail HF, Earle KA, Alila H, Pamukcu R, Moore S, Lynch RG. 2002. Rapid development of colitis in NSAID-treated IL10-deficient mice. Gastroenterology 123:1527–1542 [PubMed]
4. Boivin GP, Washington K, Yang K, Ward JM, Pretlow TP, Russell R, Besselsen DG, Godfrey VL, Doetschman T, Dove WF, Pitot HC, Halberg RB, Itzkowitz SH, Groden J, Coffey RJ. 2003. Pathology of mouse models of intestinal cancer: consensus report and recommendations. Gastroenterology 124:762–777 [PubMed]
5. Bouma G, Strober W. 2003. The immunological and genetic basis of inflammatory bowel disease. Nat Rev Immunol 3:521–533 [PubMed]
6. Burich A, Hershberg R, Waggie K, Zeng W, Brabb T, Westrich G, Viney JL, Maggio-Price L. 2001. Helicobacter-induced inflammatory bowel disease in IL10- and T cell-deficient mice. Am J Physiol Gastrointest Liver Physiol 281:G764–G778 [PubMed]
7. Comunian L, Moura S, Paglia A, Nicoli J, Guerra J, Rocha G, Queiroz D. 2006. Detection of Helicobacter species in the gastrointestinal tract of wild rodents from Brazil. Curr Microbiol 53:370–373 [PubMed]
8. Dijkstra G, Yuvaraj S, Jiang HQ, Bun JC, Moshage H, Kushnir N, Peppelenbosch MP, Cebra JJ, Bos NA. 2007. Early bacterial dependent induction of inducible nitric oxide synthase (iNOS) in epithelial cells upon transfer of CD45RB(high) CD4(+) T cells in a model for experimental colitis. Inflamm Bowel Dis 13:1467–1474 [PubMed]
9. Feng S, Ku K, Hodzic E, Lorenzana E, Freet K, Barthold SW. 2005. Differential detection of five mouse-infecting Helicobacter species by multiplex PCR. Clin Diagn Lab Immunol 12:531–536 [PMC free article] [PubMed]
10. Fox JG, Gorelick PL, Kullberg MC, Ge Z, Dewhirst FE, Ward JM. 1999. A novel urease-negative Helicobacter species associated with colitis and typhlitis in IL10-deficient mice. Infect Immun 67:1757–1762 [PMC free article] [PubMed]
11. Franklin CL, Gorelick PL, Riley LK, Dewhirst FE, Livingston RS, Ward JM, Beckwith CS, Fox JG. 2001. Helicobacter typhlonius sp. nov., a novel murine urease-negative Helicobacter species. J Clin Microbiol 39:3920–3926 [PMC free article] [PubMed]
12. Franklin CL, Riley LK, Livingston RS, Beckwith CS, Hook RR, Jr, Besch-Williford CL, Hunziker R, Gorelick PL. 1999. Enteric lesions in SCID mice infected with “Helicobacter typhlonicus,” a novel urease-negative Helicobacter species. Lab Anim Sci 49:496–505 [PubMed]
13. Ge Z, Lee A, Whary MT, Rogers AB, Maurer KJ, Taylor NS, Schauer DB, Fox JG. 2008. Helicobacter hepaticus urease is not required for intestinal colonization but promotes hepatic inflammation in male A/JCr mice. Microb Pathog 45:18–24 [PMC free article] [PubMed]
14. Hale LP, Gottfried MR, Swidsinski A. 2005. Piroxicam treatment of IL10-deficient mice enhances colonic epithelial apoptosis and mucosal exposure to intestinal bacteria. Inflamm Bowel Dis 11:1060–1069 [PubMed]
15. Hale LP, Greer PK, Trinh CT, Gottfried MR. 2005. Treatment with oral bromelain decreases colonic inflammation in the IL10-deficient murine model of inflammatory bowel disease. Clin Immunol 116:135–142 [PubMed]
16. Hale LP, Perera D, Gottfried MR, Magio-Price L, Srinivasan S, Marchuk D. 2007. Neonatal coinfection with Helicobacter species markedly accelerates the development of inflammation-associated colonic neoplasia in IL10 −/− mice. Helicobacter 12:598–604 [PubMed]
17. Johansson SK, Feinstein RE, Johansson KE, Lindberg AV. 2006. Occurrence of Helicobacter species other than H. hepaticus in laboratory mice and rats in Sweden. Comp Med 56:110–113 [PubMed]
18. Kullberg MC, Andersen JF, Gorelick PL, Caspar P, Suerbaum S, Fox JG, Cheever AW, Jankovic D, Sher A. 2003. Induction of colitis by a CD4+ T cell clone specific for a bacterial epitope. Proc Natl Acad Sci U S A 100:15830–15835 [PMC free article] [PubMed]
19. Livingston RS, Riley LK, Steffen EK, Besch-Williford CL, Hook RR, Jr, Franklin CL. 1997. Serodiagnosis of Helicobacter hepaticus infection in mice by an enzyme- linked immunosorbent assay. J Clin Microbiol 35:1236–1238 [PMC free article] [PubMed]
20. Maggio-Price L, Bielefeldt-Ohmann H, Treuting P, Iritani BM, Zeng W, Nicks A, Tsang M, Shows D, Morrissey P, Viney JL. 2005. Dual infection with Helicobacter bilis and Helicobacter hepaticus in P-glycoprotein-deficient mdr1a−/− mice results in colitis that progresses to dysplasia. Am J Pathol 166:1793–1806 [PMC free article] [PubMed]
21. Maggio-Price L, Treuting P, Zeng W, Tsang M, Bielefeldt-Ohmann H, Iritani BM. 2006. Helicobacter infection is required for inflammation and colon cancer in Smad3-deficient mice. Cancer Res 66:828–838 [PubMed]
22. Mahler M, Bedigian HG, Burgett BL, Bates RJ, Hogan ME, Sundberg JP. 1998. Comparison of four diagnostic methods for detection of Helicobacter species in laboratory mice. Lab Anim Sci 48:85–91 [PubMed]
23. Myles MH, Livingston RS, Franklin CL. 2004. Pathogenicity of Helicobacter rodentium in A/JCr and SCID mice. Comp Med 54:549–557 [PubMed]
24. National Research Council 1996. Guide for the care and use of laboratory animals. Washington (DC): National Academy Press
25. Pratt JS, Sachen KL, Wood HD, Eaton KA, Young VB. 2006. Modulation of host immune responses by the cytolethal distending toxin of Helicobacter hepaticus. Infect Immun 74:4496–4504 [PMC free article] [PubMed]
26. Rao VP, Poutahidis T, Ge Z, Nambiar PR, Boussahmain C, Wang YY, Horwitz BH, Fox JG, Erdman SE. 2006. Innate immune inflammatory response against enteric bacteria Helicobacter hepaticus induces mammary adenocarcinoma in mice. Cancer Res 66:7395–7400 [PubMed]
27. Scavizzi F, Raspa M. 2006. Helicobacter typhlonius was detected in the sex organs of three mouse strains but did not transmit vertically. Lab Anim 40:70–79 [PubMed]
28. Sharp JM, Vanderford DA, Chichlowski M, Myles MH, Hale LP. 2008. Heliobacter infection decreases reproductive success of IL10-deficient mice. Comp Med 58: 447–453 [PMC free article] [PubMed]
29. Shen Z, Fox JG, Dewhirst FE, Paster BJ, Foltz CJ, Yan L, Shames B, Perry L. 1997. Helicobacter rodentium sp. nov., a urease-negative Helicobacter species isolated from laboratory mice. Int J Syst Bacteriol 47:627–634 [PubMed]
30. Shomer NH, Dangler CA, Marini RP, Fox JG. 1998. Helicobacter bilisHelicobacter rodentium coinfection associated with diarrhea in a colony of SCID mice. Lab Anim Sci 48:455–459 [PubMed]
31. Shomer NH, Fox JG, Juedes AE, Ruddle NH. 2003. Helicobacter-induced chronic active lymphoid aggregates have characteristics of tertiary lymphoid tissue. Infect Immun 71:3572–3577 [PMC free article] [PubMed]
32. Sturlan S, Oberhuber G, Beinhauer BG, Tichy B, Kappel S, Wang J, Rogy MA. 2001. Interleukin-10-deficient mice and inflammatory bowel disease associated cancer development. Carcinogenesis 22:665–671 [PubMed]
33. Swidsinski A, Loening-Baucke V, Lochs H, Hale LP. 2005. Spatial organization of bacterial flora in normal and inflamed intestine: a fluorescence in situ hybridization study in mice. World J Gastroenterol 11:1131–1140 [PubMed]
34. Swidsinski A, Weber J, Loening-Baucke V, Hale LP, Lochs H. 2005. Spatial organization and composition of the mucosal flora in patients with inflammatory bowel disease. J Clin Microbiol 43:3380–3389 [PMC free article] [PubMed]
35. Whary MT, Danon SJ, Feng Y, Ge Z, Sundina N, Ng V, Taylor NS, Rogers AB, Fox JG. 2006. Rapid onset of ulcerative typhlocolitis in B6.129P2-IL10tm1Cgn (IL10−/−) mice infected with Helicobacter trogontum is associated with decreased colonization by altered Schaedler's flora. Infect Immun 74:6615–6623 [PMC free article] [PubMed]
36. Whary MT, Fox JG. 2004. Natural and experimental Helicobacter infections. Comp Med 54:128–158 [PubMed]
37. Young VB, Knox KA, Pratt JS, Cortez JS, Mansfield LS, Rogers AB, Fox JG, Schauer DB. 2004. In vitro and in vivo characterization of Helicobacter hepaticus cytolethal distending toxin mutants. Infect Immun 72:2521–2527 [PMC free article] [PubMed]

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