Helicobacter Typhlonius and Helicobacter Rodentium
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Comparative Medicine Vol 58, No 6 Copyright 2008 December 2008 by the American Association for Laboratory Animal Science Pages 534–541 Helicobacter typhlonius and Helicobacter rodentium Differentially Affect the Severity of Colon Inflammation and Inflammation-Associated Neoplasia in IL10-Deficient Mice Maciej Chichlowski,1 Julie M Sharp,2 Deborah A Vanderford,2 Matthew H Myles,3 and Laura P Hale1,* 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 sever- ity of IBD and incidence of inflammation-associated colonic neoplasia were assessed in the presence and absence of antiHelico- bacter 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 antiin- flammatory effect of antibiotic therapy inHelicobacter -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. Abbreviation: IBD, inflammatory bowel disease. various species of intestinal bacteria and the mucosa in mice and Inflammatory bowel disease (IBD) is hypothesized to develop humans with IBD.33,34 Those studies showed that in normal mice, due to aberrant immune responses induced by gut microbes.5 most bacterial groups are separated from the mucosal surface IBD does not occur in germ-free IL10−/− mice,2,15 indicating the by either a mucus layer that excludes bacteria or, in the cecum importance of microorganisms as environmental triggers of in- and proximal colon, by an ‘interlaced’ layer that is composed of testinal inflammation. However, conventionally colonized or tightly packed bacteria. The interlaced or mucus layer thus limits specific pathogen-free IL10−/− mice may develop colitis spontane- the contact of the bulk of the enteric bacteria with the mucosal ously2,32 or in response to specific triggers such as nonsteroidal epithelium. In contrast, complex biofilms composed of multiple antiinflammatory drugs3,14 or infections with certain bacteria.6,16,18 species of bacteria that were firmly adherent to the mucosal surface The normal lack of ongoing immune responses against bacteria were identified in the majority of colon tissue samples collected from in subjects without IBD has been attributed to the immunolog- humans and mice with IBD.33,34 The presence of a biofilm abrogates ic tolerance that specifically downregulates immune responses the protective effects of the normal layer of mucus and can allow against antigens derived from these bacteria. Nevertheless, de- luminal bacterial antigens and toxins to reach the unshielded epithe- spite a large number of studies, no single bacterial type has ful- lial surface, where they can trigger cascades of host inflammatory filled Koch postulates and been confirmed as a cause of IBD in responses. Situations that cause defects in the epithelial surface or animals or humans. degrade the protective qualities of mucus or the interlaced layer (or Previous studies used fluorescence in situ hybridization with both) may allow contact of bacterial antigens and adjuvants with im- probes specific for bacterial 16S rRNA combined with conven- mune cells located in the lamina propria and lead to the generation tional histologic techniques to study the relationships between of immune responses that result in IBD.34 Helicobacter species are used frequently to model microbial trig- Received: 01 May 2008. Revision requested: 14 Jun 2008. Accepted: 12 Jul 2008. gers of colon inflammation, because they have previously been 1Department of Pathology and 2Office of Animal Welfare Assurance, Duke University, 3 linked to the development of both IBD- and inflammation-as- Durham, North Carolina; Department of Veterinary Pathobiology, University of Missouri, 11,21,29 Columbia, Missouri. sociated neoplasia. Most studies have been performed by *Corresponding author. Email: [email protected] using Helicobacter hepaticus or H. bilis.20 However, H. typhlonius, 534 Helicobacter infection in IL10−/− mice H. rodentium, H. muridarum, H. ganmani, H. trogontum and other Helicobacter spp. All animal studies were approved by the Duke species8,12,17,29,35 can also be endemic in research animal facilities. University Institutional Animal Care and Use Committee. The pathophysiologic effects of these less-common Helicobacter Helicobacter infection. Mice were infected on day 0 with H. species are, for the most part, poorly investigated. typhlonius (clinical isolate DU-01),16 H. rodentium (MIT 95-1707; Most rodent Helicobacter species are urease-negative and there- equal to ATCC type strain 700285)29 by gavage of a single dose fore preferentially colonize the intestine, but some species pro- of 500 µl culture (approximately 5 × 107 organisms unless other- duce urease enzyme and can translocate to the liver or colonize wise specified). For inoculation, both strains were grown in Bru- the biliary system.13 H. typhlonius was shown to cause an enteric cella broth (Becton Dickinson, Franklin Lakes, NJ) as previously disease characterized by mucosal hyperplasia and associated in- described.19 Cultures were agitated with a stir bar in a 250-ml flammation in the cecum and colon in immunodeficient mice11,23 Erlenmeyer flask and were incubated for 24 h at 37 °C in a an −/− 18 and IL10 mice. H. typhlonius is genetically related most closely atmosphere of 90% N2, 5% H2, and 5% CO2. Noninfected con- to H. hepaticus, having only 2.36% difference in the 16S rRNA gene trols were sham-gavaged or received sterile broth or PBS only. sequence, but H. typhlonius has a unique intervening sequence in Because initial experiments showed frequent failure of sustained this gene that makes it easily recognizable by PCR.9,12 Molecular H. rodentium infection when H. rodentium and H. typhlonius were detection of this pathogen with PCR is rapid, sensitive and allows given simultaneously, an additional dose of H. rodentium was giv- the detection of the early phases of infection; further enhanced en on day 2 to mice in the mixed-infection group. Infection was sensitivity is achieved with nested primers.22 One of the most im- confirmed at 1 wk after infection and at 4-wk intervals thereafter portant features of PCR is that it can be performed noninvasively by analysis of feces by PCR (see PCR of Helicobacter organisms). on fecal pellets. Data regarding the pathogenetic mechanisms of Breeding to generate neonatally infected mice typically began H. rodentium are scarce.35,36 H. rodentium alone apparently does immediately after infection and was performed in triads consist- not cause hepatitis or enteritis in A/JCr or C.B-17/IcrCrl-scidBr ing of 1 male and 2 female mice. Infection in pups born to in- mice; however, coinfection with H. hepaticus and H. rodentium was fected dams was confirmed 1 to 2 wk postweaning. Mice were 1 associated with augmented cecal gene expression and clinical euthanized by CO2 asphyxiation if they developed 15% body diarrheal disease in immunodeficient mice compared with mice weight loss or rectal prolapse or when they reached 7 to 8 months infected with H. hepaticus alone.23 of age. All mice in this study were evaluated pathologically for Previous reports demonstrated that H. typhlonius was capa- both colitis and neoplasia. Sentinel mice exposed repeatedly to ble of initiating colitis in adult IL10−/− mice.10,11 In those studies, dirty bedding from the mice used in this study were negative for colitis was relatively mild, with no development of inflamma- parasites by microscopic exam, negative for Citrobacter rodentium tion-associated neoplasia. H. rodentium has been described to be by fecal culture, and negative by serology for a panel of 22 mu- nonpathogenic in adult wild-type mice but did enhance cytokine rine protozoal, bacterial, and viral pathogens, including murine production in the cecum of mice also infected with H. hepaticus.23 parvovirus, murine hepatitis virus, and murine norovirus. The We recently observed rapid onset of severe IBD and a high in- studies were performed at an AAALAC-accredited institution. cidence of inflammation-associated neoplasia in IL10−/− mice All studies were performed in accordance with The Guide for the that were coinfected with both H. typhlonius and H. rodentium Care and Use of Laboratory Animals.24 as pups.16 The current study was undertaken