Helicobacter hepaticus Cholesterol-α-glucosyltransferase is Essential for Establishing Colonization in Male A/JCr Mice

Background Helicobacter pylori cholesterol-α-glucosyltransferase (cgt) is essential for survival of H. pylori in mice. Enterohepatic H. hepaticus, the cause of colonic and hepatocellular carcinoma in susceptible mouse strains, contains an ortholog of the H. pylori cgt. However, the role of cgt in the pathogenesis of H. hepaticus has not been investigated. Materials and Methods Two cgt-deficient isogenic mutants of wild-type H. hepaticus (WT) 3B1 were generated and used to inoculate male A/JCr mice. Cecal and hepatic colonization levels of the mutants and WT 3B1 as well as select inflammation-associated cytokines were measured by qPCR at 4 months postinoculation. Results Both mutants were undetectable in the cecum of any inoculated mice (10 per mutant) but were detected in two livers (one for each mutant); by contrast, 9 and 7 of 10 mice inoculated with WT 3B1 were qPCR positive in the ceca and livers, respectively. The mice inoculated with the mutants developed significantly less severe hepatic inflammation (p < .05) and also produced significantly lower hepatic mRNA levels of proinflammatory cytokines Ifn-γ (p < .01) and Tnf-α (p ≤ .02) as well as anti-inflammatory factors Il10 and Foxp3 compared with the WT 3B1-inoculated mice. Additionally, the WT 3B1-inoculated mice developed significantly higher Th1-associated IgG2a (p < .0001) and Th2-associated IgG1 responses (p < .0001) to H. hepaticus infection than mice dosed with isogenic cgt mutants. Conclusion Our data indicate that the cholesterol-α-glucosyltransferase is required for establishing colonization of the intestine and liver and therefore plays a critical role in the pathogenesis of H. hepaticus.National Institutes of Health (U.S.) (Grant R010D011141)National Institutes of Health (U.S.) (Grant 01CA026731)National Institutes of Health (U.S.) (Grant R01AT004326)National Institutes of Health (U.S.) (Grant P30-ES002109

Serologic Evidence that Ascaris and Toxoplasma Infections Impact Inflammatory Responses to Helicobacter pylori in Colombians

Background: Helicobacter pylori-infected children from coastal Tumaco, Colombia, have more parasitism, and adults have lower gastric cancer risk compared with high-altitude Pasto/Tuquerres residents. Because helminth and Toxoplasma gondii infections alter helicobacter gastritis in rodent models, we determined whether seropositivity to Ascaris lumbricoides or T. gondii was associated with Th2-IgG1 or Th1-IgG2 responses to H. pylori. Methods: Sera (240) from the two populations were evaluated for A. lumbricoides and T. gondii seropositivity and results correlated with IgE and IgG isotype responses to H. pylori. Results: Most Tumaco children and adults were seropositive for A. lumbricoides (89%, 66%), T. gondii (59%, 98%), or both (45%, 66%). In contrast, seropositivity among Pasto/Tuquerres children was much lower (9%A. lumbricoides, 11%T. gondii, and 2% dual positive) but increased in adults (58%A. lumbricoides, 82%T. gondii, and 41% dual positive). A. lumbricoides seropositivity correlated with elevated IgE and anti-inflammatory Th2-IgG1 responses to H. pylori, while T. gondiigondii seropositivity was linked to elevated IgE, pro-inflammatory Th1-IgG2, IgG3, and IgG4 responses to H. pylori. Individuals with high T. gondii titers had reduced Th1-IgG2, IgG3, and IgG4 responses to H. pylori. Conclusions: Results support regional differences for childhood parasitism and indicate A. lumbricoides and T. gondii infections may impact inflammatory responses to H. pylori and partially explain differences in gastric cancer risk in Colombia.National Institutes of Health (U.S.) (Grant T32RR07036)National Institutes of Health (U.S.) (Grant PO1CA028842)National Institutes of Health (U.S.) (Grant P30ES02109

Helicobacter hepaticus infection in mice: models for understanding lower bowel inflammation and cancer

Pioneering work in the 1990s first linked a novel microaerobic bacterium, Helicobacter hepaticus, with chronic active hepatitis and inflammatory bowel disease in several murine models. Targeted H. hepaticus infection experiments subsequently demonstrated its ability to induce colitis, colorectal cancer, and extraintestinal diseases in a number of mouse strains with defects in immune function and/or regulation. H. hepaticus is now widely utilized as a model system to dissect how intestinal microbiota interact with the host to produce both inflammatory and tolerogenic responses. This model has been used to make important advances in understanding factors that regulate both acquired and innate immune response within the intestine. Further, it has been an effective tool to help define the function of regulatory T cells, including their ability to directly inhibit the innate inflammatory response to gut microbiota. The complete genomic sequence of H. hepaticus has advanced the identification of several virulence factors and aided in the elucidation of H. hepaticus pathogenesis. Delineating targets of H. hepaticus virulence factors could facilitate novel approaches to treating microbially induced lower bowel inflammatory diseases.National Institutes of Health (U.S.) (grant R01-DK052413)National Institutes of Health (U.S.) (grant P01-CA026731)National Institutes of Health (U.S.) (grant R01-CA067529)National Institutes of Health (U.S.) (grant P30-ES02109)National Institutes of Health (U.S.) (grant R01-A1052267)National Institutes of Health (U.S.) (grantR01-CA108854

Helminth co-infection in Helicobacter pylori infected INS-GAS mice attenuates gastric premalignant lesions of epithelial dysplasia and glandular atrophy and preserves colonization resistance of the stomach to lower bowel microbiota

Higher prevalence of helminth infections in Helicobacter pylori infected children was suggested to potentially lower the life-time risk for gastric adenocarcinoma. In rodent models, helminth co-infection does not reduce Helicobacter-induced inflammation but delays progression of pre-malignant gastric lesions. Because gastric cancer in INS-GAS mice is promoted by intestinal microflora, the impact of Heligmosomoides polygyrus co-infection on H. pylori-associated gastric lesions and microflora were evaluated. Male INS-GAS mice co-infected with H. pylori and H. polygyrus for 5 months were assessed for gastrointestinal lesions, inflammation-related mRNA expression, FoxP3[superscript +] cells, epithelial proliferation, and gastric colonization with H. pylori and Altered Schaedler Flora. Despite similar gastric inflammation and high levels of proinflammatory mRNA, helminth co-infection increased FoxP3[superscript +] cells in the corpus and reduced H. pylori-associated gastric atrophy (p < 0.04), dysplasia (p < 0.02) and prevented H. pylori-induced changes in the gastric flora (p < 0.05). This is the first evidence of helminth infection reducing H. pylori-induced gastric lesions while inhibiting changes in gastric flora, consistent with prior observations that gastric colonization with enteric microbiota accelerated gastric lesions in INS-GAS mice. Identifying how helminths reduce gastric premalignant lesions and impact bacterial colonization of the H. pylori infected stomach could lead to new treatment strategies to inhibit progression from chronic gastritis to cancer in humans.RO1-CA67529R01DK052413PO1CA26731P01 CA028842P30ESO2109R01DK06507

Impaired cholecystokinin-induced gallbladder emptying incriminated in spontaneous “black” pigment gallstone formation in germfree Swiss Webster mice

“Black” pigment gallstones form in sterile gallbladder bile in the presence of excess bilirubin conjugates (“hyperbilirubinbilia”) from ineffective erythropoiesis, hemolysis, or induced enterohepatic cycling (EHC) of unconjugated bilirubin. Impaired gallbladder motility is a less well-studied risk factor. We evaluated the spontaneous occurrence of gallstones in adult germfree (GF) and conventionally housed specific pathogen-free (SPF) Swiss Webster (SW) mice. GF SW mice were more likely to have gallstones than SPF SW mice, with 75% and 23% prevalence, respectively. In GF SW mice, gallstones were observed predominately in heavier, older females. Gallbladders of GF SW mice were markedly enlarged, contained sterile black gallstones composed of calcium bilirubinate and <1% cholesterol, and had low-grade inflammation, edema, and epithelial hyperplasia. Hemograms were normal, but serum cholesterol was elevated in GF compared with SPF SW mice, and serum glucose levels were positively related to increasing age. Aged GF and SPF SW mice had deficits in gallbladder smooth muscle activity. In response to cholecystokinin (CCK), gallbladders of fasted GF SW mice showed impaired emptying (females: 29%; males: 1% emptying), whereas SPF SW females and males emptied 89% and 53% of volume, respectively. Bilirubin secretion rates of GF SW mice were not greater than SPF SW mice, repudiating an induced EHC. Gallstones likely developed in GF SW mice because of gallbladder hypomotility, enabled by features of GF physiology, including decreased intestinal CCK concentration and delayed intestinal transit, as well as an apparent genetic predisposition of the SW stock. GF SW mice may provide a valuable model to study gallbladder stasis as a cause of black pigment gallstones.National Institutes of Health (U.S.) (Training Grant T32-OD10978-26)National Institutes of Health (U.S.) (Training Grant P30-ES002109)Kinship Foundation. Searle Scholars Progra

Gastric colonisation with a restricted commensal microbiota replicates the promotion of neoplastic lesions by diverse intestinal microbiota in the Helicobacter pylori INS-GAS mouse model of gastric carcinogenesis

Objectives: Gastric colonisation with intestinal flora (IF) has been shown to promote Helicobacter pylori (Hp)-associated gastric cancer. However, it is unknown if the mechanism involves colonisation with specific or diverse microbiota secondary to gastric atrophy. Design: Gastric colonisation with Altered Schaedler's flora (ASF) and Hp were correlated with pathology, immune responses and mRNA expression for proinflammatory and cancer-related genes in germ-free (GF), Hp monoassociated (mHp), restricted ASF (rASF; 3 species), and specific pathogen-free (complex IF), hypergastrinemic INS-GAS mice 7 months postinfection. Results: Male mice cocolonised with rASFHp or IFHp developed the most severe pathology. IFHp males had the highest inflammatory responses, and 40% developed invasive gastrointestinal intraepithelial neoplasia (GIN). Notably, rASFHp colonisation was highest in males and 23% developed invasive GIN with elevated expression of inflammatory biomarkers. Lesions were less severe in females and none developed GIN. Gastritis in male rASFHp mice was accompanied by decreased Clostridum species ASF356 and Bacteroides species ASF519 colonisation and an overgrowth of Lactobacillus murinus ASF361, supporting that inflammation-driven atrophy alters the gastric niche for GI commensals. Hp colonisation also elevated expression of IL-11 and cancer-related genes, Ptger4 and Tgf-β, further supporting that Hp infection accelerates gastric cancer development in INS-GAS mice. Conclusions: rASFHp colonisation was sufficient for GIN development in males, and lower GIN incidence in females was associated with lower inflammatory responses and gastric commensal and Hp colonisation. Colonisation efficiency of commensals appears more important than microbial diversity and lessens the probability that specific gastrointestinal pathogens are contributing to cancer risk.National Institutes of Health (U.S.) (grant R01 AI37750)National Institutes of Health (U.S.) (grant R01 CA093405)National Institutes of Health (U.S.) (grant P30-ES02109)National Institutes of Health (U.S.) (grant P01 CA028842)National Institutes of Health (U.S.) (grant T32 RR07036

An Analysis of the Role of the Indigenous Microbiota in Cholesterol Gallstone Pathogenesis

Background and Aims: Cholesterol gallstone disease is a complex process involving both genetic and environmental variables. No information exists regarding what role if any the indigenous gastrointestinal microbiota may play in cholesterol gallstone pathogenesis and whether variations in the microbiota can alter cholesterol gallstone prevalence rates. Methods: Genetically related substrains (BALB/cJ and BALB/cJBomTac) and (BALB/AnNTac and BALB/cByJ) of mice obtained from different vendors were compared for cholesterol gallstone prevalence after being fed a lithogenic diet for 8 weeks. The indigenous microbiome was altered in these substrains by oral gavage of fecal slurries as adults, by cross-fostering to mice with divergent flora at <1day of age or by rederiving into a germ-free state. Results: Alterations in the indigenous microbiome altered significantly the accumulation of mucin gel and normalized gallbladder weight but did not alter cholesterol gallstone susceptibility in conventionally housed SPF mice. Germ-free rederivation rendered mice more susceptible to cholesterol gallstone formation. This susceptibility appeared to be largely due to alterations in gallbladder size and gallbladder wall inflammation. Colonization of germ-free mice with members of altered Schaedler flora normalized the gallstone phenotype to a level similar to conventionally housed mice. Conclusions: These data demonstrate that alterations in the gastrointestinal microbiome may alter aspects of cholesterol gallstone pathogenesis and that in the appropriate circumstances these changes may impact cholesterol cholelithogenesis.National Institutes of Health (U.S.) (Grant T32OD010978)National Institutes of Health (U.S.) (Grant P30ES002109)National Institutes of Health (U.S.) (Grant R01AT004326

Helicobacter cinaedi Induced Typhlocolitis in Rag-2-Deficient Mice

Background Helicobacter cinaedi, an enterohepatic helicobacter species (EHS), is an important human pathogen and is associated with a wide range of diseases, especially in immunocompromised patients. It has been convincingly demonstrated that innate immune response to certain pathogenic enteric bacteria is sufficient to initiate colitis and colon carcinogenesis in recombinase-activating gene (Rag)-2-deficient mice model. To better understand the mechanisms of human IBD and its association with development of colon cancer, we investigated whether H. cinaedi could induce pathological changes noted with murine enterohepatic helicobacter infections in the Rag2[superscript −/−] mouse model. Materials and Methods Sixty 129SvEv Rag2[superscript −/−] mice mouse were experimentally or sham infected orally with H. cinaedi strain CCUG 18818. Gastrointestinal pathology and immune responses in infected and control mice were analyzed at 3, 6 and 9 months postinfection (MPI). H. cinaedi colonized the cecum, colon, and stomach in infected mice. Results H. cinaedi induced typhlocolitis in Rag2[superscript −/−] mice by 3 MPI and intestinal lesions became more severe by 9 MPI. H. cinaedi was also associated with the elevation of proinflammatory cytokines, interferon-γ, tumor-necrosis factor-α, IL-1β, IL-10; iNOS mRNA levels were also upregulated in the cecum of infected mice. However, changes in IL-4, IL-6, Cox-2, and c-myc mRNA expressions were not detected. Conclusions Our results indicated that the Rag2[superscript −/−] mouse model will be useful to continue investigating the pathogenicity of H. cinaedi, and to study the association of host immune responses in IBD caused by EHS.United States. National Institutes of Health (R01-0D011141)United States. National Institutes of Health (R01-CA067529)United States. National Institutes of Health (P30-ES002109)United States. National Institutes of Health (P01-CA026731

Intestinal Microbiota Composition of Interleukin-10 Deficient C57BL/6J Mice and Susceptibility to Helicobacter hepaticus-Induced Colitis

The mouse pathobiont Helicobacter hepaticus can induce typhlocolitis in interleukin-10-deficient mice, and H. hepaticus infection of immunodeficient mice is widely used as a model to study the role of pathogens and commensal bacteria in the pathogenesis of inflammatory bowel disease. C57BL/6J Il10[superscript −/−] mice kept under specific pathogen-free conditions in two different facilities (MHH and MIT), displayed strong differences with respect to their susceptibilities to H. hepaticus-induced intestinal pathology. Mice at MIT developed robust typhlocolitis after infection with H. hepaticus, while mice at MHH developed no significant pathology after infection with the same H. hepaticus strain. We hypothesized that the intestinal microbiota might be responsible for these differences and therefore performed high resolution analysis of the intestinal microbiota composition in uninfected mice from the two facilities by deep sequencing of partial 16S rRNA amplicons. The microbiota composition differed markedly between mice from both facilities. Significant differences were also detected between two groups of MHH mice born in different years. Of the 119 operational taxonomic units (OTUs) that occurred in at least half the cecum or colon samples of at least one mouse group, 24 were only found in MIT mice, and another 13 OTUs could only be found in MHH samples. While most of the MHH-specific OTUs could only be identified to class or family level, the MIT-specific set contained OTUs identified to genus or species level, including the opportunistic pathogen, Bilophila wadsworthia. The susceptibility to H. hepaticus-induced colitis differed considerably between Il10[superscript −/−] mice originating from the two institutions. This was associated with significant differences in microbiota composition, highlighting the importance of characterizing the intestinal microbiome when studying murine models of IBD.National Institutes of Health (U.S.) (Grant NIH P01-CA26731)National Institutes of Health (U.S.) (Grant NIH P30ES0026731)National Institutes of Health (U.S.) (Grant NIH R01-OD011141