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Hepatocytes injury caused by cytolethal distending toxin (CDT) are major events during helicobacter hepaticus (H.hepaticus) infection. Recent study showed that pre-survival autophagy was promoted against CdtB subunit induced DNA damage. In the present study, we demonstrated that inflammatory cytokines IL-6, IL-1β, TNF-α, IFN-α, IFN-γ expression and STAT phosphorylation were promoted by CdtB. Besides, CdtB decreased cell viability while promote apoptosis in mouse liver (AML12) cells. Especially, apoptotic protein caspase-9, caspase-3 and PARP were activated while the ratio of Bcl-2/Bax was decreased after CdtB treatment. Moreover, apoptosis induced by CdtB was inhibited due to Erk/p38 MAPK signaling pathway suppression performed with SB203580 or U0126. Meanwhile, we found that CdtB increased autophagic marker levels accompanied by Akt/mTOR/P70S6K signaling pathway in a dose dependent manner. To assess the correlation between autophagy and apoptosis induced by H.hepaticus, chloroquine (CQ, 50 μM) was employed to inhibit autophagy. The result showed that inhibition of autophagy with CQ treatment promoted apoptosis induced by CdtB. Altogether, all these results suggest that CdtB triggers apoptosis via MAPK/Erk/p38 signaling pathway in caspase dependent manner, which was prevented by autophagy in AML12 cells. Collectively, our findings provide new insights into the virulence potential of CdtB on the molecular pathogenesis throughout H.hepaticus infection., Competing Interests: Declaration of competing interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2022 Elsevier Inc. All rights reserved.)

Gut microbiota and the immune system are in constant exchange shaping both host immunity and microbial communities. Here, improper immune regulation can cause inflammatory bowel disease (IBD) and colitis. Antibody therapies blocking signaling through the CD40-CD40L axis showed promising results as these molecules are deregulated in certain IBD patients. To better understand the mechanism, we used transgenic DC-LMP1/CD40 animals with a constitutive CD40-signal in CD11c + cells, causing a lack of intestinal CD103 + dendritic cells (DCs) and failure to induce regulatory T (iTreg) cells. These mice rapidly develop spontaneous fatal colitis, accompanied by dysbiosis and increased inflammatory IL-17 + IFN-γ + Th17/Th1 and IFN-γ + Th1 cells. In the present study, we analyzed the impact of the microbiota on disease development and detected elevated IgA- and IgG-levels in sera from DC-LMP1/CD40 animals. Their serum antibodies specifically bound intestinal bacteria, and by proteome analysis, we identified a 60 kDa chaperonin GroEL (Hsp60) from Helicobacter hepaticus ( Hh ) as the main specific antigen targeted in the absence of iTregs. When re-derived to a different Hh -free specific-pathogen-free (SPF) microbiota, mice showed few signs of disease, normal microbiota, and no fatality. Upon recolonization of mice with Hh , the disease developed rapidly. Thus, the present work identifies GroEL/Hsp60 as a major Hh -antigen and its role in disease onset, progression, and outcome in this colitis model. Our results highlight the importance of CD103 + DC- and iTreg-mediated immune tolerance to specific pathobionts to maintain healthy intestinal balance.

The prevalence of gastric Helicobacter pylori (Hp) infection is ~50% of the world population. However, how Hp infection influences inflammatory bowel disease in humans is not fully defined. In this study, we examined whether co-infection with Hp influenced Helicobacter hepaticus (Hh)-induced intestinal pathology in Rag2 -/- mice. Rag2 -/- mice of both sexes were infected with Hh, of which a subgroup was followed by infection with Hp two weeks later. Co-infected males, but not females, had significantly higher total colitis index scores in the colon at both 10 and 21 weeks post-Hh infection (WPI) and developed more severe dysplasia at 21 WPI compared with mono-Hh males. There were no significant differences in colonization levels of gastric Hp and colonic Hh between sexes or time-points. In addition, mRNA levels of colonic Il-1β , Ifnγ , Tnfα , Il-17A , Il-17F , Il-18 , and Il-23 , which play important roles in the development and function of proinflammatory innate lymphoid cell groups 1 and 3, were significantly up-regulated in the dually infected males compared with mono-Hh males at 21 WPI. These data suggest that concomitant Hp infection enhances the inflammatory responses in the colon of-Hh-infected Rag2 -/- males, which results in more severe colitis and dysplasia.

It has been documented that Helicobacter hepaticus (H. hepaticus) infection is linked to hepatic inflammation and fibrosis. Interleukin 33 (IL-33) is a cytokine involved in inflammatory and fibrotic diseases, but its relevance to H. hepaticus infection-induced liver inflammation and fibrosis is unknown. In this study, we found that the expression of IL-33 in mice liver was significantly induced by H. hepaticus infection at 24 weeks post infection (WPI). Immunohistochemistry analysis revealed that IL-33 was transferred from the nucleus to the cytoplasm due to infection. The quantitation of inflammatory cytokine and histopathology evaluation showed that IL-33 knockdown attenuated the H. hepaticus-induced hepatic inflammation and fibrosis. More importantly, H. hepaticus promoted the expression of the IL-33 receptor ST2 on cell surfaces, and the expression of ST2 then activated the expression nuclear factor-κB (p65), α-SMA, and Erk1/2. These observations provide novel insights into the pathogenic mechanism of hepatic inflammation and fibrosis during H. hepaticus infection., Competing Interests: Declaration of competing interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2020 Elsevier Inc. All rights reserved.)

Inflammatory bowel disease and colonic tumors induced by Helicobacter hepaticus (Hh) infection in susceptible mouse strains are utilized to dissect the mechanisms underlying similar human diseases. In our study, infection with genotoxic cytolethal distending toxin-producing Hh in 129/SvEv Rag2 -/- Il10 -/- gpt delta (RagIl10gpt) mice of both sexes for 21 weeks induced significantly more severe cecal and colonic pathology compared to uninfected controls. The mutation frequencies in the infected RagIl10gpt males were 2.1-fold higher for the cecum and 1.7-fold higher for the colon than male RagIl10gpt controls. In addition, there was a 12.5-fold increase of G:C-to-T:A transversions in the colon of Hh-infected males compared to controls. In contrast, there was no statistical significance in mutation frequencies between infected female Rag2Il10gpt mice and controls. Moreover, Hh infection in RagIl10gpt males significantly up-regulated transcription of Tnfα and iNos, and decreased mRNA levels of cecal Atm compared to the infected females; there was no significant difference in mRNA levels of Il-22, Il-17A, Ifnγ and Atr between the infected males and females. Significantly higher levels of cecal and colonic iNos expression and γH2AX-positive epithelial cells (a biomarker for double-strand DNA breaks [DSB]) in Hh-infected Rag2Il10gpt males vs. Hh-infected females were noted. Finally, Hh infection and associated inflammation increased levels of intestinal mucosa-associated genotoxic colibactin-producing pks+ Escherichia coli. Elevated Tnfα and iNos responses and bacterial genotoxins, in concert with suppression of the DSB repair responses, may have promoted mutagenesis in the lower bowel mucosa of Hh-infected male RagIl10gpt mice., (© 2019 UICC.)

Study Question: Does the genotype of the surrogate mother modulate the body composition and immunity of her offspring?, Summary Answer: C57BL/6J (B6) progenies carried by immunodeficient NOD SCID (NS) mothers had increased adaptive but decreased innate, immune responsiveness in comparison with the same genotype offspring carried by immunocompetent mothers, B6 and BALB/c (C); the B6 progenies carried by the same genotype mothers also showed higher body fat than the others., What Is Known Already: Differences in the major histocompatibility complex (MHC) genes between mother and foetus is considered as an important factor in prenatal embryo development, whereas the impact of such dissimilarity on the phenotype of the mature progeny is unclear., Study Design, Size, Duration: Transplantation of two-cell mouse embryos into recipient females of the different MHC (H2) genotypes was used as an approach to simulate three variants of the immunogenic mother-foetus interaction: (i) bidirectional immunogenic dialogue between B6 (H2b haplotype) embryos and C (H2d haplotype) surrogate mother; (ii) one-way immunogenic interaction between B6 embryos and immunodeficient NS (H2g7 haplotype) surrogate mother and (iii) reduced immunogenetic dialogue between embryos and surrogate mother of the same H2b haplotype resulting in only a maternal response to HY antigens of male foetuses. Delivered by Caesarean section, pups were fostered by lactating B6 females and weighed after weaning (n = 171). Body mass and composition and innate and adaptive immunity were assessed in selected progeny groups at 9-11 weeks of age., Participants/materials, Setting, Methods: The study was performed on the specific pathogen-free mouse, inbred strains C57BL/6J, NOD SCID and BALB/c. Plasma progesterone in pregnant females was measured by enzyme-linked immunosorbent assay (ELISA). Body composition was determined by magnetic resonance spectroscopy using a low-field NMR spectrometer (EchoMRI, USA). To assess peritoneal macrophage responses (innate immunity) to anthrax, lactate dehydrogenase (LDH) and interleukin-1 (IL-1β) were measured in a culture medium 24 h after the addition of both anthrax-lethal factor and anthrax-protective antigen. To assess adaptive immunity, 9-10 males in experimental groups were infected with Helicobacter hepaticus. Faeces collected 2 and 4 weeks after infection was used for quantitative assessment of the H. hepaticus DNA by real-time polymerase chain reaction. IgA, interferon (IFN-γ), tumour necrosis factor (TNFα), interleukin-17 (IL-17) and interleukin-10 (IL-10) in colon tissue and IgG in serum were determined in samples collected 4 weeks after gavage with H. hepaticus using ELISA. For statistical analyses, ANCOVA, post hoc least significant difference (LSD) test, Student's t-test, Spearman rank correlations and χ2 test were performed. P-value <0.05 was considered as a statistically significant difference., Main Results and the Role of Chance: ANCOVA with litter size and age as covariates revealed significant effects of the surrogate mother genotype on body mass and percent of fat in their adult progeny (F2149 = 15.60, P < 0.001 and F2149 = 5.02, P = 0.007, respectively). Adult B6 mice carried by B6 surrogate mothers were characterized by a higher percentage of body fat in comparison with offspring that were carried by NS and C females. In comparison with the male offspring carried by the B6 and C mothers, male B6 progenies carried by immunodeficient NS mothers had a higher humoral immune response (serum IgG) against oral infection with H. hepaticus, but lower in vitro macrophage IL-1β reaction to the anthrax. Four weeks after the infection of offspring, concentrations of serum IgG and colon IL-10 correlated positively with maternal progesterone on Day 4 after embryo transfer and negatively with DNA of H. hepaticus. One-way ANOVA confirmed a statistically significant impact of surrogate mother genotype on adaptive (IgG) and innate (IL-1β) immunity (F2.26 = 26.39, P < 0.001 and F2.27 = 5.89, P = 0.008, respectively)., Large Scale Data: N/A., Limitations, Reasons for Caution: The main limitation of our study is the number of combinations of mother and foetus interactions, in particular, transfer of only one embryo genotype was used. Also, it is a descriptive study, which requires further analysis of the epigenetic mechanisms of the observed phenotypic effects of surrogate mother genotype., Wider Implications of the Findings: Our experimental data demonstrate that the transfer of inbred embryos to surrogate mothers of the different genotypes is a prospective experimental model for the study of epigenetic effects of the immunogenetic interactions between mother and foetus. The experimental approach tested in our study will be in demand for the development of criteria for choosing surrogate mothers. In particular, immunocompetence of the surrogate mother along with genetic distance of her MHC alleles to the transferred embryos have a significant impact on offspring development., Study Funding/competing Interest(s): This work was supported by the Russian FPI (6/099/2017), budget projects (0324-2016-0002 and 0324-2018-0016) and implemented using the equipment of the Centre for Genetic Resources of Laboratory Animals at ICG SB RAS, supported by the Ministry of Education and Science of Russia (Unique project identifier RFMEFI62117X0015). The authors report no conflicts of interest.

Both microbial and host genetic factors contribute to the pathogenesis of autoimmune diseases. There is accumulating evidence that microbial species that potentiate chronic inflammation, as in inflammatory bowel disease, often also colonize healthy individuals. These microorganisms, including the Helicobacter species, can induce pathogenic T cells and are collectively referred to as pathobionts. However, how such T cells are constrained in healthy individuals is not yet understood. Here we report that host tolerance to a potentially pathogenic bacterium, Helicobacter hepaticus, is mediated by the induction of RORγt + FOXP3 + regulatory T (iT reg ) cells that selectively restrain pro-inflammatory T helper 17 (T H 17) cells and whose function is dependent on the transcription factor c-MAF. Whereas colonization of wild-type mice by H. hepaticus promoted differentiation of RORγt-expressing microorganism-specific iT reg cells in the large intestine, in disease-susceptible IL-10-deficient mice, there was instead expansion of colitogenic T H 17 cells. Inactivation of c-MAF in the T reg cell compartment impaired differentiation and function, including IL-10 production, of bacteria-specific iT reg cells, and resulted in the accumulation of H. hepaticus-specific inflammatory T H 17 cells and spontaneous colitis. By contrast, RORγt inactivation in T reg cells had only a minor effect on the bacteria-specific T reg and T H 17 cell balance, and did not result in inflammation. Our results suggest that pathobiont-dependent inflammatory bowel disease is driven by microbiota-reactive T cells that have escaped this c-MAF-dependent mechanism of iT reg -T H 17 homeostasis.

Genome-wide association studies have linked polymorphisms in the autophagy gene ATG16L1 with susceptibility to inflammatory bowel disease (IBD). However, the cell-type-specific effects of autophagy on the regulation of chronic intestinal inflammation have not been investigated. Here, we assessed the effect of myeloid-specific or intestinal epithelial cell (IEC)-specific deletion of Atg16l1 on chronic colitis triggered by the intestinal opportunistic pathogen Helicobacter hepaticus in mice. Although Atg16l1 deficiency in myeloid cells had little effect on disease, mice selectively lacking Atg16l1 in IECs (Atg16l1 VC ) developed severely exacerbated pathology, accompanied by elevated pro-inflammatory cytokine secretion and increased IEC apoptosis. Using ex vivo IEC organoids, we demonstrate that autophagy intrinsically controls TNF-induced apoptosis and in vivo blockade of TNF attenuated the exacerbated pathology in Atg16l1 VC mice. These findings suggest that the IBD susceptibility gene ATG16L1 and the process of autophagy within the epithelium control inflammation-induced apoptosis and barrier integrity to limit chronic intestinal inflammation., (Copyright © 2017 Elsevier Inc. All rights reserved.)

Cells of the monocyte/macrophage lineage play important roles in the pathogenesis of inflammatory bowel diseases, but they are also present in the normal healthy intestine, where they are critical for maintaining homeostasis. It has been unclear whether the proinflammatory roles of intestinal macrophages reflect altered behavior of the existing resident cells, or whether they involve recruitment of a distinct cell type. Here, we have explored these ideas using the model of colitis induced by Helicobacter hepaticus in the context of neutralization or deletion of interleukin-10 (IL-10). Granulocytes and monocytes made up most of the inflammatory myeloid infiltrates found in the colon of H. hepaticus -infected colitic mice, rising to a peak within 2 weeks of H. hepaticus inoculation but taking several months to resolve completely. The inflammatory response was dependent on the combined presence of H. hepaticus and absence of IL-10 and was accompanied by increased production of inflammatory mediators such as IL-1β, tumor necrosis factor alpha (TNF-α), IL-6, and IL-23p19 by infiltrating myeloid cells, mostly relatively immature cells of the macrophage lineage that express intermediate levels of CX3CR1. In contrast, the population of mature CX3CR1 hi macrophages did not expand as markedly during colitis, and these cells made little contribution to inflammatory mediator production. Taking into account their numerical dominance in the myeloid compartment, we conclude that newly recruited monocytes are the main source of proinflammatory mediators in colitis induced in the absence of IL-10 signaling and that altered behavior of mature macrophages is not a major component of this pathology., (Copyright © 2018 Bain et al.)

Multiple pathogenic Gram-negative bacteria produce the cytolethal distending toxin (CDT) with activity of DNase I; CDT can induce DNA double-strand breaks (DSBs), G2/M cell cycle arrest, and apoptosis in cultured mammalian cells. However, the link of CDT to in vivo tumorigenesis is not fully understood. In this study, 129/SvEv Rag2 -/- mice were gavaged with wild-type Helicobacter hepatics 3B1(Hh) and its isogenic cdtB mutant HhcdtBm7, followed by infection for 10 and 20 weeks (WPI). HhCDT deficiency did not affect cecal colonization levels of HhcdtBm7, but attenuated severity of cecal pathology in HhcdtBm7-infected mice. Of importance, preneoplasic dysplasia was progressed to cancer from 10 to 20 WPI in the Hh-infected mice but not in the HhcdtBm7-infected mice. In addition, the loss of HhCDT significantly dampened transcriptional upregulation of cecal Tnfα and Il-6, but elevated Il-10 mRNA levels when compared to Hh at 10 WPI. Furthermore, the presence of HhCDT increased numbers of lower bowel intestinal γH2AX-positive epithelial cells (a marker of DSBs) at both 10 and 20 WPI and augmented phospho-Stat3 foci + intestinal crypts (activation of Stat3) at 20 WPI. Our findings suggest that CDT promoted Hh carcinogenesis by enhancing DSBs and activation of the Tnfα/Il-6-Stat3 signaling pathway., (© 2017 John Wiley & Sons Ltd.)

Although IL-10 promotes a regulatory phenotype of CD11c+ dendritic cells and macrophages in vitro, the role of IL-10 signaling in CD11c+ cells to maintain intestinal tolerance in vivo remains elusive. To this aim, we generated mice with a CD11c-specific deletion of the IL-10 receptor alpha (Cd11ccreIl10rafl/fl). In contrast to the colon, the small intestine of Cd11ccreIl10rafl/fl mice exhibited spontaneous crypt hyperplasia, increased numbers of intraepithelial lymphocytes and lamina propria T cells, associated with elevated levels of T cell-derived IFNγ and IL-17A. Whereas naive mucosal T-cell priming was not affected and oral tolerance to ovalbumin was intact, augmented T-cell function in the lamina propria was associated with elevated numbers of locally dividing T cells, expression of T-cell attracting chemokines and reduced T-cell apoptosis. Upon stimulation, intestinal IL-10Rα deficient CD11c+ cells exhibited increased activation associated with enhanced IL-6 and TNFα production. Following colonization with Helicobacter hepaticus Cd11ccreIl10rafl/fl mice developed severe large intestinal inflammation characterized by infiltrating T cells and increased levels of Il17a, Ifng, and Il12p40. Altogether these findings demonstrate a critical role of IL-10 signaling in CD11c+ cells to control small intestinal immune homeostasis by limiting reactivation of local memory T cells and to protect against Helicobacter hepaticus-induced colitis., Competing Interests: The authors declare no conflicts of interests.

In space, the lifestyle, relative sterility of spaceship and extreme environmental stresses, such as microgravity and cosmic radiation, can compromise the balance between human body and human microbiome. An astronaut's body during spaceflight encounters increased risk for microbial infections and conditions because of immune dysregulation and altered microbiome, i.e. dysbiosis. This risk is further heightened by increase in virulence of pathogens in microgravity. Health status of astronauts might potentially benefit from maintaining a healthy microbiome by specifically managing their diet on space in addition to probiotic therapies. This review focuses on the current knowledge/understanding of how spaceflight affects human immunity and microbiome.

IL-1 family members are central mediators of host defense. In this article, we show that the novel IL-1 family member IL-36γ was expressed during experimental colitis and human inflammatory bowel disease. Germ-free mice failed to induce IL-36γ in response to dextran sodium sulfate (DSS)-induced damage, suggesting that gut microbiota are involved in its induction. Surprisingly, IL-36R-deficient (Il1rl2(-/-)) mice exhibited defective recovery following DSS-induced damage and impaired closure of colonic mucosal biopsy wounds, which coincided with impaired neutrophil accumulation in the wound bed. Failure of Il1rl2(-/-) mice to recover from DSS-induced damage was associated with a profound reduction in IL-22 expression, particularly by colonic neutrophils. Defective recovery of Il1rl2(-/-) mice could be rescued by an aryl hydrocarbon receptor agonist, which was sufficient to restore IL-22 expression and promote full recovery from DSS-induced damage. These findings implicate the IL-36/IL-36R axis in the resolution of intestinal mucosal wounds., (Copyright © 2015 by The American Association of Immunologists, Inc.)

Adenosine is a purine metabolite that can mediate anti-inflammatory responses in the digestive tract through the A(2A) adenosine receptor (A(2A)AR). We examined the role of this receptor in the control of inflammation in the adoptive transfer model of colitis. Infection of A(2A)AR(-/-) mice with Helicobacter hepaticus increased colonic inflammation scores compared with uninfected A(2A)AR controls. Comparison of T cell subsets in wild-type and A(2A)AR(-/-) mice revealed differences in markers associated with activated helper T (Th) cells and regulatory T (Treg) cells. Previous studies showed that expression of A(2A)AR on CD45RB(HI) and CD45RB(LO) Th cells is essential for the proper regulation of colonic inflammation. Adoptive transfer of CD45RB(HI) with CD45RB(LO) from wild-type mice into RAG1(-/-)/A(2A)AR(-/-) mice induced severe disease within 3 wk, although transfer of the same subsets into RAG1(-/-) mice does not induce colitis. This suggests that the presence of A(2A)AR on recipient cells is also important for controlling colitis. To investigate the role of A(2A)AR in myeloid cells, chimeric recipients were generated by injection of bone marrow from RAG1(-/-) or RAG1(-/-)/A(2A)AR(-/-) mice into irradiated RAG1(-/-) mice. After adoptive transfer, these recipients did not develop colitis, regardless of A(2A)AR expression by the donor. Together, our results suggest that the control of inflammation in vivo is dependent on A(2A)AR signaling through multiple cell types that collaborate in the regulation of colitis by responding to extracellular adenosine., (Copyright © 2014 the American Physiological Society.)

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(-/-) 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(-/-) 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.

Background: Patients with inflammatory bowel disease (IBD) are at increase risk for bone loss and fractures. Therefore, in the present study, we examined the effect of experimental IBD on bone health., Methods: We used a murine model of colitis, Helicobacter hepaticus-infected interleukin-10-deficient animals. Molecular and histological properties of bone and intestine were examined to identify the immunopathological consequences of colitis in male and female mice., Results: At 6 weeks postinfection, we observed significant trabecular bone loss in male mice but surprisingly not in female mice. This was true for both distal femur and vertebral locations. In addition, H. hepaticus infection suppressed osteoblast markers only in male mice. Consistent with effects on bone health, male mice with H. hepaticus infection had more severe colitis as determined by histology and elevated levels of inflammatory cytokines in the colon. Although H. hepaticus levels in the stool appeared similar in male and female mice 1 week after infection, by 6 weeks, H. hepaticus levels were greater in male mice, indicating that H. hepaticus survival and virulence within the gastrointestinal tract could be gender dependent., Conclusion: In summary, H. hepaticus-induced colitis severity and associated bone loss is gender regulated, possibly as a result of gender-specific effects on H. hepaticus colonization in the mouse gastrointestinal tract and the consequent immunopathological responses.

The enterohepatic Epsilonproteobacterium Helicobacter hepaticus persistently colonizes the intestine of mice and causes chronic inflammatory symptoms in susceptible mouse strains. The bacterial factors causing intestinal inflammation are poorly characterized. A large genomic pathogenicity island, HHGI1, which encodes components of a type VI secretion system (T6SS), was previously shown to contribute to the colitogenic potential of H. hepaticus. We have now characterized the T6SS components Hcp, VgrG1, VgrG2 and VgrG3, encoded on HHGI1, including the potential impact of the T6SS on intestinal inflammation in a mouse T-cell transfer model. The H. hepaticus T6SS components were expressed during the infection and secreted in a T6SS-dependent manner, when the bacteria were cultured either in the presence or in the absence of mouse intestinal epithelial cells. Mutants deficient in VgrG1 displayed a significantly lower colitogenic potential in T-cell-transferred C57BL/6 Rag2(-/-) mice, despite an unaltered ability to colonize mice persistently. Intestinal microbiota analyses demonstrated only minor changes in mice infected with wild-typeH. hepaticus as compared with mice infected with VgrG1-deficient isogenic bacteria. In addition, competitive assays between both wild-type and T6SS-deficient H. hepaticus, and between wild-type H. hepaticus and Campylobacter jejuni or Enterobacteriaceae species did not show an effect of the T6SS on interbacterial competitiveness. Therefore, we suggest that microbiota alterations did not play a major role in the changes of pro-inflammatory potential mediated by the T6SS. Cellular innate pro-inflammatory responses were increased by the secreted T6SS proteins VgrG1 and VgrG2. We therefore concluded that the type VI secretion component VgrG1 can modulate and specifically exacerbate the innate pro-inflammatory effect of the chronic H. hepaticus infection., (© 2012 John Wiley & Sons Ltd.)

Background: The heterogeneity of hepatitis C virus (HCV) infection cannot always be explained by HCV genotypes or host genetic factors, raising the issue of possible cofactors. A new form of hepatitis leading to liver cancer was discovered in 1992 in mice, owing to an infection by Helicobacter hepaticus. Moreover, several studies showed an association between the presence of HCV and Helicobacter in the liver of patients with severe liver diseases suggesting a possible synergism between the two pathogens. In an HCV transgenic mouse model with a B6C3F1 background, the combination of H. hepaticus infection and the HCV transgene resulted in a significantly greater incidence and multiplicity of preneoplastic and neoplastic liver foci in males., Objectives: Because the mouse genetic background is a major determinant in the development of liver disease, our aim was to test the synergism between HCV and H. hepaticus infection using transgenic mice with a more sensitive genetic background to H. hepaticus infection., Methods: For this purpose, four groups of mice were followed up to 14 months, the presence of H. hepaticus was monitored by PCR and hepatic lesions were looked for., Results: We found that H. hepaticus, but not the HCV transgene, increased the number of hepatic lesions. The presence of carcinoma was more likely to occur on a background of hepatitis, and the overall lesions were more frequent in the presence of steatosis. The effect of the mouse genetic background was greater than the effect of the HCV transgene and was sufficient to promote lesions particularly via its sensitivity to H. hepaticus infection., Conclusions: Genetic susceptibility may be a more important factor than expected. Indeed, the synergism between HCV and H. hepaticus infection involved in liver disease may be highly host dependent., (© 2012 Blackwell Publishing Ltd.)

Although very high levels of interleukin (IL)-1β are present in the intestines of patients suffering from inflammatory bowel diseases (IBD), little is known about the contribution of IL-1β to intestinal pathology. Here, we used two complementary models of chronic intestinal inflammation to address the role of IL-1β in driving innate and adaptive pathology in the intestine. We show that IL-1β promotes innate immune pathology in Helicobacter hepaticus-triggered intestinal inflammation by augmenting the recruitment of granulocytes and the accumulation and activation of innate lymphoid cells (ILCs). Using a T cell transfer colitis model, we demonstrate a key role for T cell-specific IL-1 receptor (IL-1R) signals in the accumulation and survival of pathogenic CD4(+) T cells in the colon. Furthermore, we show that IL-1β promotes Th17 responses from CD4(+) T cells and ILCs in the intestine, and we describe synergistic interactions between IL-1β and IL-23 signals that sustain innate and adaptive inflammatory responses in the gut. These data identify multiple mechanisms through which IL-1β promotes intestinal pathology and suggest that targeting IL-1β may represent a useful therapeutic approach in IBD.

Cistanche deserticola has commonly been used in traditional Chinese medicine to treat many health problems including irritable bowel syndrome or constipation. This study was designed to test the efficacy of a water-extract of C. deserticola in the prevention of colorectal cancer in a mouse model. Polysaccharide-rich water-extract of C. deserticola was prepared by boiling its stem powder in distilled water. Tgfb1Rag2 null mice were used as an experimental model. Here we showed that feeding of water-extract of C. deserticola significantly reduced the number of mucosal hyperplasia and intestinal helicobacter infection in mice. This beneficial effect correlated with significant stimulation of the immune system, evidenced by the enlargement of the spleens with increased number of splenic macrophage and natural killer cells, and with more potent cytotoxicity of splenocytes. In vitro water-extract of C. deserticola enhanced the cytotoxicity of naïve splenocytes against a human colon cancer cell line, and in macrophage cultures up-regulated nitric oxide synthase II expression and stimulated phagocytosis. In conclusion, our data indicate that oral administration of C. deserticola extract reduces inflammatory hyperplastic polyps and helicobacter infection in mice by its immune-stimulatory activity, suggesting that C. deserticola extract may have potential in preventing intestinal inflammation disorders including colorectal cancer., (Copyright © 2011 John Wiley & Sons, Ltd.)

Aim: To identify and characterize drosophila mothers against decapentaplegic (SMAD)3-dependent changes in immune cell populations following infection with Helicobacter hepaticus (H. hepaticus)., Methods: SMAD3(-/-) (n = 19) and colitis-resistant SMAD3(+/-) (n = 24) mice (8-10 wk of age) were infected with H. hepaticus and changes in immune cell populations [T lymphocytes, natural killer (NK) cells, T regulatory cells] were measured in the spleen and mesenteric lymph nodes (MsLNs) at 0 d, 3 d, 7 d and 28 d post-infection using flow cytometry. Genotype-dependent changes in T lymphocytes and granzyme B(+) cells were also assessed after 28 d in proximal colon tissue using immunohistochemistry., Results: As previously observed, SMAD3(-/-), but not SMAD3(+/-) mice, developed colitis, peaking at 4 wk post-infection. No significant changes in T cell subsets were observed in the spleen or in the MsLNs between genotypes at any time point. However, CD4(+) and CD8(+)/CD62L(lo) cells, an effector T lymphocyte population, as well as NK cells (NKp46/DX5(+)) were significantly higher in the MsLNs of SMAD3(-/-) mice at 7 d and 28 d post-infection. In the colon, a higher number of CD3(+) cells were present in SMAD3(-/-) compared to SMAD3(+/-) mice at baseline, which did not significantly change during infection. However, the number of granzyme B(+) cells, a marker of cytolytic lymphocytes, significantly increased in SMAD3(-/-) mice 28 d post-infection compared to both SMAD3(+/-) mice and to baseline values. This was consistent with more severe colitis development in these animals., Conclusion: Data suggest that defects in SMAD3 signaling increase susceptibility to H. hepaticus-induced colitis through aberrant activation and/or dysregulation of effector lymphocytes.

To investigate how different enterohepatic Helicobacter species (EHS) influence Helicobacter pylori gastric pathology, C57BL/6 mice were infected with Helicobacter hepaticus or Helicobacter muridarum, followed by H. pylori infection 2 weeks later. Compared to H. pylori-infected mice, mice infected with H. muridarum and H. pylori (HmHp mice) developed significantly lower histopathologic activity index (HAI) scores (P < 0.0001) at 6 and 11 months postinoculation (MPI). However, mice infected with H. hepaticus and H. pylori (HhHp mice) developed more severe gastric pathology at 6 MPI (P = 0.01), with a HAI at 11 MPI (P = 0.8) similar to that of H. pylori-infected mice. H. muridarum-mediated attenuation of gastritis in coinfected mice was associated with significant downregulation of proinflammatory Th1 (interlukin-1beta [Il-1β], gamma interferon [Ifn-γ], and tumor necrosis factor-alpha [Tnf-α]) cytokines at both time points and Th17 (Il-17A) cytokine mRNA levels at 6 MPI in murine stomachs compared to those of H. pylori-infected mice (P < 0.01). Coinfection with H. hepaticus also suppressed H. pylori-induced elevation of gastric Th1 cytokines Ifn-γ and Tnf-α (P < 0.0001) but increased Th17 cytokine mRNA levels (P = 0.028) at 6 MPI. Furthermore, mRNA levels of Il-17A were positively correlated with the severity of helicobacter-induced gastric pathology (HhHp>H. pylori>HmHp) (at 6 MPI, r² = 0.92, P < 0.0001; at 11 MPI, r² = 0.82, P < 0.002). Despite disparate effects on gastritis, colonization levels of gastric H. pylori were increased in HhHp mice (at 6 MPI) and HmHp mice (at both time points) compared to those in mono-H. pylori-infected mice. These data suggest that despite consistent downregulation of Th1 responses, EHS coinfection either attenuated or promoted the severity of H. pylori-induced gastric pathology in C57BL/6 mice. This modulation was related to the variable effects of EHS on gastric interleukin 17 (IL-17) responses to H. pylori infection.

To model inflammatory bowel disease, we assessed infection with Helicobacter hepaticus 3B1 (ATCC 51449) and a potential probiotic Lactobacillus reuteri (ATCC PTA-6475) in gnotobiotic B6.129P2-IL-10(tm1Cgn) (IL-10(-/-) ) mice. No typhlocolitis developed in germ-free controls (n=21) or in L. reuteri (n=8) or H. hepaticus (n=18) mono-associated mice for 20 weeks post-infection. As positive controls, three specific pathogen-free IL-10(-/-) mice dosed with H. hepaticus developed severe typhlocolitis within 11 weeks. Because L. reuteri PTA-6475 has anti-inflammatory properties in vitro, it was unexpected to observe significant typhlocolitis (P<0·0001) in mice that had been infected with L. reuteri followed in 1 week by H. hepaticus (n=16). The H. hepaticus colonization was not affected through 20 weeks post-infection but L. reuteri colonization was lower in co-infected compared with L. reuteri mono-associated mice at 8-11 weeks post-infection (P<0·05). Typhlocolitis was associated with an increased T helper type 1 serum IgG2c response to H. hepaticus in co-infected mice compared with H. hepaticus mono-associated mice (P<0·005) and similarly, mRNA expression in caecal-colonic tissue was elevated at least twofold for chemokine ligands and pro-inflammatory interleukin-1α (IL-1α), IL-1β, IL-12 receptor, tumour necrosis factor-α and inducible nitric oxide synthase. Anti-inflammatory transforming growth factor-β, lactotransferrin, peptidoglycan recognition proteins, Toll-like receptors 4, 6, 8 and particularly 9 gene expression, were also elevated only in co-infected mice (P<0·05). These data support that the development of typhlocolitis in H. hepaticus-infected IL-10(-/-) mice required co-colonization with other microbiota and in this study, required only L. reuteri. Although the effects other microbiota may have on H. hepaticus virulence properties remain speculative, further investigations using this gnotobiotic model are now possible., (© 2011 The Authors. Immunology © 2011 Blackwell Publishing Ltd.)

Gut microbes play a major role in carcinogenesis of the gastrointestinal tract. We and others have shown in mouse models that colonic bacteria also influence the development of extraintestinal cancers including hepatocellular and mammary carcinomas. Microbes such as Helicobacter hepaticus invoke a proinflammatory microenvironment in the lower bowel that may extend to distant organs, often in the absence of histologically evident inflammation. Innate immunity plays a crucial role in the promotion of liver cancer and other systemic diseases by gut microbes. Additional mechanisms include type 1 adaptive immunity, altered metabolism, and oxidative stress. Emerging links between host genetics, gut microbes, inflammatory bowel disease and colorectal cancer also may prove useful for the correlation of specific bacterial populations with extraintestinal neoplasms. Interruption of deleterious host-microbe networks through judicious use of antibiotics and targeted molecular therapies may help reduce the incidence of liver, breast, and other human cancers., (© 2011 Landes Bioscience)

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.

Background & Aims: Toll-like receptors (TLR) are innate immune receptors involved in recognition of the intestinal microflora; they are expressed by numerous cell types in the intestine, including epithelial cells, myeloid cells, and lymphocytes. Little is known about the relative contributions of TLR signaling in distinct cellular compartments to intestinal homeostasis. We aimed to define the roles of TLR signals in distinct cell types in the induction and regulation of chronic intestinal inflammation., Methods: We assessed the roles of the shared TLR signaling adaptor protein, MyD88, in several complementary mouse models of inflammatory bowel disease, mediated by either innate or adaptive immune activation. MyD88-deficient mice and bone marrow chimeras were used to disrupt TLR signals selectively in distinct cellular compartments in the intestine., Results: MyD88-dependent activation of myeloid cells was required for the development of chronic intestinal inflammation. By contrast, although epithelial cell MyD88 signals were required for host survival, they were insufficient to induce intestinal inflammation in the absence of an MyD88-competent myeloid compartment. MyD88 expression by T cells was not required for their pathogenic and regulatory functions in the intestine., Conclusions: Cellular compartmentalization of MyD88 signals in the intestine allow the maintenance of host defense and prevent deleterious inflammatory responses., (Copyright (c) 2010 AGA Institute. Published by Elsevier Inc. All rights reserved.)

The key role of interleukin (IL)-23 in the pathogenesis of autoimmune and chronic inflammatory disorders is supported by the identification of IL-23 receptor (IL-23R) susceptibility alleles associated with inflammatory bowel disease, psoriasis and ankylosing spondylitis. IL-23-driven inflammation has primarily been linked to the actions of T-helper type 17 (TH17) cells. Somewhat overlooked, IL-23 also has inflammatory effects on innate immune cells and can drive T-cell-independent colitis. However, the downstream cellular and molecular pathways involved in this innate intestinal inflammatory response are poorly characterized. Here we show that bacteria-driven innate colitis is associated with an increased production of IL-17 and interferon-gamma in the colon. Stimulation of colonic leukocytes with IL-23 induced the production of IL-17 and interferon-gamma exclusively by innate lymphoid cells expressing Thy1, stem cell antigen 1 (SCA-1), retinoic-acid-related orphan receptor (ROR)-gammat and IL-23R, and these cells markedly accumulated in the inflamed colon. IL-23-responsive innate intestinal cells are also a feature of T-cell-dependent models of colitis. The transcription factor ROR-gammat, which controls IL-23R expression, has a functional role, because Rag-/-Rorc-/- mice failed to develop innate colitis. Last, depletion of Thy1+ innate lymphoid cells completely abrogated acute and chronic innate colitis. These results identify a previously unrecognized IL-23-responsive innate lymphoid population that mediates intestinal immune pathology and may therefore represent a target in inflammatory bowel disease.

The gastrointestinal tract harbors a diverse microbiota that has coevolved with mammalian hosts. Though most associations are symbiotic or commensal, some resident bacteria (termed pathobionts) have the potential to cause disease. Bacterial type VI secretion systems (T6SSs) are one mechanism for forging host-microbial interactions. Here we reveal a protective role for the T6SS of Helicobacter hepaticus, a Gram-negative bacterium of the intestinal microbiota. H. hepaticus mutants with a defective T6SS display increased numbers within intestinal epithelial cells (IECs) and during intestinal colonization. Remarkably, the T6SS directs an anti-inflammatory gene expression profile in IECs, and CD4+ T cells from mice colonized with T6SS mutants produce increased interleukin-17 in response to IECs presenting H. hepaticus antigens. Thus, the H. hepaticus T6SS limits colonization and intestinal inflammation, promoting a balanced relationship with the host. We propose that disruption of such balances contributes to human disorders such as inflammatory bowel disease and colon cancer., (Copyright 2010 Elsevier Inc. All rights reserved.)

Activities of CD4(+) regulatory (T(REG)) cells restore immune homeostasis during chronic inflammatory disorders. Roles for T(REG) cells in inflammation-associated cancers, however, are paradoxical. It is widely believed that T(REG) function in cancer mainly to suppress protective anticancer responses. However, we demonstrate here that T(REG) cells also function to reduce cancer risk throughout the body by efficiently downregulating inflammation arising from the gastrointestinal (GI) tract. Building on a "hygiene hypothesis" model in which GI infections lead to changes in T(REG) that reduce immune-mediated diseases, here we show that gut bacteria-triggered T(REG) may function to inhibit cancer even in extraintestinal sites. Ability of bacteria-stimulated T(REG) to suppress cancer depends on interleukin (IL)-10, which serves to maintain immune homeostasis within bowel and support a protective antiinflammatory T(REG) phenotype. However, under proinflammatory conditions, T(REG) may fail to provide antiinflammatory protection and instead contribute to a T helper (Th)-17-driven procarcinogenic process; a cancer state that is reversible by downregulation of inflammation. Consequently, hygienic individuals with a weakened IL-10 and T(REG)-mediated inhibitory loop are highly susceptible to the carcinogenic consequences of elevated IL-6 and IL-17 and show more frequent inflammation-associated cancers. Taken together, these data unify seemingly divergent disease processes such as autoimmunity and cancer and help explain the paradox of T(REG) and inflammation in cancer. Enhancing protective T(REG) functions may promote healthful longevity and significantly reduce risk of cancer.

Background: Helicobacter hepaticus, the prototype for enterohepatic Helicobacter species, colonizes the lower intestinal and hepatobiliary tracts of mice and causes typhlocolitis, hepatitis, and hepatocellular carcinoma in susceptible mouse strains. Cytolethal distending toxin (CDT) is the only known virulence factor found in H. hepaticus. CDT of several Gram-negative bacteria is associated with double-stranded DNA breaks resulting in cell cycle arrest and death of a wide range of eukaryotic cells in vitro. We previously observed H. hepaticus CDT (HhCDT) mediated apoptosis in INT407 cells. However, the exact mechanism for the induction of the apoptotic pathway by HhCDT is unknown. The objective of this study was to identify the apoptotic signaling pathway induced by HhCDT in INT407 cells., Materials and Methods: INT407 cells were incubated with or without recombinant HhCDT for 0-72 hours. H2AX phosphorylation and apoptotic parameters were analyzed., Results: H2AX was phosphorylated 24 hours postexposure to HhCDT. Expression of pro-apoptotic Bax protein was upregulated after 24 hours, while Bcl(2) expression decreased. Cytochrome c was released from mitochondria after 12-24 hours of exposure. Concurrently, caspase 3/7 and 9 were activated. However, pretreatment of INT407 cells with caspase inhibitor (Z-VAD-FMK) inhibited the activation of caspase 3/7 and 9. Significant activity of caspase 8 was not observed in toxin treated cells. Activation of caspase 3/7 and caspase 9 confirms the involvement of the mitochondrial apoptotic pathway in HhCDT-treated cells., Conclusion: These findings show, for the first time, the ability of HhCDT to induce apoptosis via the mitochondrial pathway.

Infection of mice with Helicobacter hepaticus is common in research colonies, yet little is known about how this persistent infection affects immunologic research. The goal of this study was to determine whether H. hepaticus infection status can modulate immune responses specific to herpes simplex virus type 1 (HSV1) and the phenotypic and functional characteristics of dendritic cells (DC) of mice. We compared virus-specific antibody and T cell-mediated responses in H. hepaticus-infected and noninfected mice that were inoculated intranasally with HSV1. The effect of H. hepaticus on the HSV1-specific antibody and T cell-mediated immune responses in superficial cervical and tracheobronchal lymph nodes (LN) did not reach statistical significance. Surface expression of the maturation-associated markers CD40, CD80, CD86, and MHC II and percentages of IL12p40- and TNFalpha-producing DC from spleen and colic LN in H. hepaticus-infected mice and noninfected mice were measured in separate experiments. Expression of CD40, CD86, and MHC II and percentages of IL12p40- and TNFalpha-producing DC from colic LN were decreased in H. hepaticus-infected mice. In contrast, H. hepaticus infection did not reduce the expression of these molecules by splenic DC. Expression of CD40, CD80, CD86, and MHC II on splenic DC from H. hepaticus-infected mice was increased after in vitro lipopolysaccharide stimulation. These results indicate that H. hepaticus infection can influence the results of immunologic assays in mice and support the use of H. hepaticus-free mice in immunologic research.

Background: Although severe hepatitis and liver tumors occur in a high percentage of A/J male mice naturally infected with Helicobacter hepaticus, these effects have not been observed after injection of adult mice with the bacteria., Objectives: We tested the hypothesis that perinatal exposure to the bacteria is required for liver tumorigenesis., Methods: A/J female mice were infected by intragastric (ig) or intraperitoneal (ip) treatment with 1.5 x 10(8) H. hepaticus before pregnancy. We examined offspring at progressive time intervals, including some kept until natural death in old age. A/J, BALB/c, and C57BL/6 weanling male mice were similarly treated ig with the bacteria and observed for up to 2 years., Results: After ip bacterial infection of A/J females, 41% of their male offspring developed hepatitis and 33% had hepatocellular tumors, including 18% with hepatocellular carcinoma. Treatment by the ig route resulted in a similar incidence of hepatitis in offspring (35%) but fewer total liver tumors (8%) and carcinomas (4%). By contrast, ig instillation of H. hepaticus in weanling A/J, C57BL/6, or BALB/c mice resulted in low incidence of hepatitis (0-20%) and few liver tumors, despite presence of bacteria confirmed in feces., Conclusions: Results indicate that a high incidence of liver tumors in mice infected with H. hepaticus requires perinatal exposure. Contributing perinatal factors could include known high sensitivity of neonatal liver to tumor initiation, and/or modulation of immune response to the bacterium or its toxins. Mechanisms of human perinatal sensitivity to such phenomena can be studied with this model.

Urease activity contributes to bacterial survival in the acidic environment of the stomach and is essential for persistent infection by known gastric helicobacters such as the human pathogen Helicobacter pylori. Several enterohepatic Helicobacter species (EHS) that primarily infect the less acidic intestine also have very active urease enzymes. The importance of urease and its contribution to pathogenesis for these EHS are poorly understood. In this study, we generated a urease-deficient, isogenic mutant (HhureNT9) of Helicobacter hepaticus 3B1 (Hh 3B1), an EHS that possesses a urease gene cluster similar to that of H. pylori. Lack of urease activity did not affect the level of cecal colonization by HhureNT9 compared to Hh 3B1 in male A/JCr mice (P=0.48) at 4 months post-inoculation (MPI). In contrast, there was no HhureNT9 detected in the livers of any infected mice, whereas all livers from the Hh 3B1-infected mice were PCR-positive for Hh 3B1. The mice infected with HhureNT9 developed significantly less severe hepatitis (P=0.017) and also produced significantly lower hepatic mRNA levels of proinflammatory cytokines IFN-gamma (P=0.0007) and TNF-alpha (P<0.0001) compared to the Hh 3B1-infected mice. The Hh 3B1-infected mice developed significantly higher total IgG, Th1-associated IgG2a and Th2-associated IgG1 responses to infection. These results indicate that H. hepaticus urease activity plays a crucial role in hepatic disease but is not required for cecal colonization by H. hepaticus.

Helicobacter hepaticus is a gram-negative, spiral-shaped microaerophilic bacterium associated with chronic intestinal infection leading to hepatitis and colonic and hepatic carcinomas in susceptible strains of mice. In the closely related human pathogen Helicobacter pylori, L-proline is a preferred respiratory substrate and is found at significantly high levels in the gastric juice of infected patients. A previous study of the proline catabolic PutA flavoenzymes from H. pylori and H. hepaticus revealed that Helicobacter PutA generates reactive oxygen species during proline oxidation by transferring electrons from reduced flavin to molecular oxygen. We further explored the preference for proline as a respiratory substrate and the potential impact of proline metabolism on the redox environment in Helicobacter species during host infection by disrupting the putA gene in H. hepaticus. The resulting putA knockout mutant strain was characterized by oxidative stress analysis and mouse infection studies. The putA mutant strain of H. hepaticus exhibited increased proline levels and resistance to oxidative stress relative to that of the wild-type strain, consistent with proline's role as an antioxidant. The significant increase in stress resistance was attributed to higher proline content, as no upregulation of antioxidant genes was observed for the putA mutant strain. The wild-type and putA mutant H. hepaticus strains displayed similar levels of infection in mice, but in mice challenged with the putA mutant strain, significantly reduced inflammation was observed, suggesting a role for proline metabolism in H. hepaticus pathogenicity in vivo.

Helicobacter hepaticus strain 3B1 (H. hepaticus) contains a genomic island of approximately 71 kb, HHGI1, with some of the common features shared among known bacterial pathogenicity islands. In this study, we characterized the pathogenic potential of HHGI1 by infecting B6.129-IL10 tm1Cgn (IL10-/-) mice with an isogenic mutant (namely HhPAId1) lacking 19 predicted genes within HHGI1. In contrast to H. hepaticus (P<0.001), HhPAId1 did not cause typhlocolitis and hyperplasia in IL10-/- mice. Colonization levels of HhPAId1 were significantly higher in the cecum (P<0.007) and similar in the colon (P=0.27) when compared to H. hepaticus by 13 or 16 weeks post inoculation (WPI). The magnitude of the Th1-associated IgG2c response against HhPAId1 was less than that against H. hepaticus (P<0.004). There was no significant difference in Th2-associated IgG1 responses against these two strains. Cecal and colonic mRNA levels of proinflammatory cytokines IFN-gamma, TNF-alpha and IL-17a in the HhPAId1-infected mice were significantly lower than those in the H. hepaticus-infected mice (P<0.05) at 13 WPI. These results demonstrate that genes in the HHGI1 contribute to the pathogenicity of H. hepaticus, at least in part via up-regulation of proinflammatory mediators IFN-gamma, TNF-alpha and IL-17a.

Humans are colonized by multitudes of commensal organisms representing members of five of the six kingdoms of life; however, our gastrointestinal tract provides residence to both beneficial and potentially pathogenic microorganisms. Imbalances in the composition of the bacterial microbiota, known as dysbiosis, are postulated to be a major factor in human disorders such as inflammatory bowel disease. We report here that the prominent human symbiont Bacteroides fragilis protects animals from experimental colitis induced by Helicobacter hepaticus, a commensal bacterium with pathogenic potential. This beneficial activity requires a single microbial molecule (polysaccharide A, PSA). In animals harbouring B. fragilis not expressing PSA, H. hepaticus colonization leads to disease and pro-inflammatory cytokine production in colonic tissues. Purified PSA administered to animals is required to suppress pro-inflammatory interleukin-17 production by intestinal immune cells and also inhibits in vitro reactions in cell cultures. Furthermore, PSA protects from inflammatory disease through a functional requirement for interleukin-10-producing CD4+ T cells. These results show that molecules of the bacterial microbiota can mediate the critical balance between health and disease. Harnessing the immunomodulatory capacity of symbiosis factors such as PSA might potentially provide therapeutics for human inflammatory disorders on the basis of entirely novel biological principles.

The BALB/c-IL10 null mouse strain develops colitis and colitis-associated adenocarcinomas, and is a model for idiopathic inflammatory bowel disease. We tested the hypotheses that (i) azoxymethane (AOM), a carcinogen that targets the colon, synergizes with the colonic inflammation inherent in the BALB/c-IL10 null mouse resulting in an increase in incidence, multiplicity and/or progression of AOM-induced tumors or colitis-associated adenocarcinomas; and (ii) prior infection with Helicobacter hepaticus, a common enterohepatic bacterial pathogen in many research mouse colonies, increases the incidence, multiplicity and/or progression of AOM-induced colon tumors or colitis-associated adenocarcinomas in the BALB/c-IL10 null mouse. We show that, within the timeframe examined, AOM-induced colon tumors in the BALB/c-IL10 null mouse were grossly and microscopically similar in appearance to AOM-induced colon tumors in the wild type BALB/cJ mouse. No colitis-associated adenocarcinomas were identified. Infection with H. hepaticus prior to AOM-treatment also did not result in colitis-associated adenocarcinomas but did result in a significant increase in the incidence of AOM-induced colon tumors relative to AOM treatment alone. The AOM-induced adenomas were predominantly exophytic and nodular or polypoid and localized to the distal colon. These results suggest that H. hepaticus promotes AOM-induced tumorigenesis in the BALB/c-IL10 null mouse., ((c) 2007 Wiley-Liss, Inc.)

Enterohepatic Helicobacter species infect the intestinal tracts and biliary trees of various mammals, including mice and humans, and are associated with chronic inflammatory diseases of the intestine, gallstone formation, and malignant transformation. The recent analysis of the whole genome sequence of the mouse enterohepatic species Helicobacter hepaticus allowed us to perform a functional analysis of bacterial factors that may play a role in these diseases. We tested the hypothesis that H. hepaticus suppresses or evades innate immune responses of mouse intestinal epithelial cells, which allows this pathogen to induce or contribute to chronic inflammatory disease. We demonstrated in the present study that the innate immune responses of intestinal epithelial cells to lipopolysaccharide (LPS) via Toll-like receptor 4 (TLR4) and to flagellin-mediated activation via TLR5 are reduced by H. hepaticus infection through soluble bacterial factors. In particular, H. hepaticus lysate and the soluble component LPS antagonized TLR4- and TLR5-mediated immune responses of intestinal epithelial cells. H. hepaticus lysate and LPS inhibited development of endotoxin tolerance to Escherichia coli LPS. Suppression of innate immune responses by H. hepaticus LPS thus may affect intestinal responses to the resident microbial flora, epithelial homeostasis, and intestinal inflammatory conditions.

Persistent murine infection with Helicobacter hepaticus leads to chronic gastrointestinal inflammation and neoplasia in susceptible strains. To determine the role of the virulence factor cytolethal distending toxin (CDT) in the pathogenesis of this organism, interleukin-10-deficient (IL-10-/-) mice were experimentally infected with wild-type H. hepaticus and a CDT-deficient isogenic mutant. Both wild-type H. hepaticus and the CDT-deficient mutant successfully colonized IL-10-/- mice, and they reached similar tissue levels by 6 weeks after infection. Only animals infected with wild-type type H. hepaticus developed significant typhlocolitis. However, by 4 months after infection, the CDT-deficient mutant was no longer detectable in IL-10-/- mice, whereas wild-type H. hepaticus persisted for the 8-month duration of the experiment. Animals infected with wild-type H. hepaticus exhibited severe typhlocolitis at 8 months after infection, while animals originally challenged with the CDT-deficient mutant had minimal cecal inflammation at this time point. In follow-up experiments, animals that cleared infection with the CDT-deficient mutant were protected from rechallenge with either mutant or wild-type H. hepaticus. Animals infected with wild-type H. hepaticus developed serum immunoglobulin G1 (IgG1) and IgG2c responses against H. hepaticus, while animals challenged with the CDT-deficient mutant developed significantly lower IgG2c responses and failed to mount IgG1 responses against H. hepaticus. These results suggest that CDT plays a key immunomodulatory role that allows persistence of H. hepaticus and that in IL-10-/- mice this alteration of the host immune response results in the development of colitis.

The distribution and colonization levels of the altered Schaedler flora (ASF) in their natural hosts are poorly understood. Intestinal colonization levels of the eight ASF strains in outbred Swiss Webster mice with or without Helicobacter hepaticus infection were characterized by real-time quantitative PCR. All ASF strains were detected in the cecum and colon, but some strains displayed significant variation in colonization levels with host age, gender, and H. hepaticus infection status.

Helicobacter hepaticus is an enterohepatic Helicobacter species that induces lower bowel inflammation in susceptible mouse strains, including those lacking the p50/p105 subunit of NF-kappaB. H. hepaticus-induced colitis is associated with elevated levels of IL-12 p40 expression, and p50/p105-deficient macrophages express higher levels of IL-12 p40 than wild-type macrophages after challenge with H. hepaticus. However, the molecular mechanisms by which the p50/p105 subunit of NF-kappaB suppresses IL-12 p40 expression have not yet been elucidated. In this study we have demonstrated that H. hepaticus challenge of macrophages induces ERK activation, and this event plays a critical role in inhibiting the ability of H. hepaticus to induce IL-12 p40. Activation of ERK requires both p50/p105 and the MAPK kinase kinase, Tpl-2. Inhibition of the induction of IL-12 p40 by ERK was independent of c-Rel, a known positive regulator of IL-12 p40. Instead, it was linked to the induction of c-Fos, a known inhibitor of IL-12 p40 expression. These results suggest that H. hepaticus induces ERK activation by a pathway dependent upon Tpl-2 and p105, and that activation of ERK inhibits the expression of IL-12 p40 by inducing c-Fos. Thus, a defect in ERK activation could play a pivotal role in the superinduction of IL-12 p40 observed after challenge of macrophages lacking the p50/p105 subunit of NF-kappaB with H. hepaticus.

A 70-kb genomic island (HHGI1) in Helicobacter hepaticus strain ATCC 51449 is a putative pathogenicity island (PAI). To determine the in vivo relevance of this PAI, we inoculated A/JCr mice with one of three strains of H. hepaticus: type strain Hh3B1, which contains the complete PAI, and strains HhNET and HhG, which lack all or large parts of HHGI1, respectively. Mice infected with HhG and HhNET developed less-severe hepatitis than male A/JCr mice infected with Hh3B1.

Establishment of mucosal and/or luminal colonization is the first step in the pathogenesis of many gastrointestinal bacterial pathogens. The pathogen must be able to establish itself in the face of competition from the complex microbial community that is already in place. We used culture-independent methods to monitor the colonization of the cecal mucosa of Helicobacter-free mice following experimental infection with the pathogen Helicobacter hepaticus. Two days after infection, H. hepaticus comprised a minor component of the mucosa-associated microbiota, but within 14 days, it became the dominant member of the community. Colonization of the mucosa by H. hepaticus was associated with a decrease in the overall diversity of the microbial community, in large part due to changes in evenness resulting from the relative dominance of H. hepaticus as a member of the community. Our results demonstrate that invasion of the complex gastrointestinal microbial community by a pathogenic microorganism causes reproducible and significant disturbances in the community structure. The use of non-culture-based methods to monitor these changes should lead to a greater understanding of the ecological principles that govern pathogen invasion and may lead to novel methods for the prevention and control of gastrointestinal pathogens.

Helicobacter hepaticus, which induces chronic hepatitis and typhlocolitis in susceptible mouse strains, produces a cytolethal distending toxin (CDT) consisting of CdtA, CdtB, and CdtC. A cdtB-deficient H. hepaticus isogenic mutant (HhcdtBm7) was generated and characterized for colonization parameters in four intestinal regions (jejunum, ileum, cecum, and colon) of outbred Swiss Webster (SW) mice. Inactivation of the cdtB gene abolished the ability of HhcdtBm7 to colonize female mice at both 8 and 16 weeks postinfection (wpi), whereas HhcdtBm7 colonized all of four intestinal regions of three of five males at 8 wpi and then was eliminated by 16 wpi. Wild-type (WT) H. hepaticus was detected in the corresponding intestinal regions of both male and female mice at 8 and 16 wpi; however, colonization levels of WT H. hepaticus in the cecum and colon of male mice were approximately 1,000-fold higher than in females (P < 0.0079) at 16 wpi. Infection with WT H. hepaticus, but not HhcdtBm7, at 8 wpi was associated with significantly increased mRNA level of ileal and cecal gamma interferon (IFN-gamma) in females (P < 0.016 and 0.031 between WT H. hepaticus-infected and sham-dosed females, respectively). In contrast, the mRNA levels of IFN-gamma were significantly higher in the colon (P < 0.0079) and trended to be higher in the cecum (P < 0.15) in the HhcdtBm7-colonized male mice versus the sham-dosed controls at 8 wpi. In addition, mRNA levels of ileal IFN-gamma were significantly higher in the control females than males at 8 wpi (P < 0.016). There were significantly higher Th1-associated immunoglobulin G2a (IgG2a), Th2-associated IgG1 and mucosal IgA (P < 0.002, 0.002, 0.002, respectively) responses in the mice infected with WT H. hepaticus when compared to HhcdtBm7 at 16 wpi. Colonic interleukin-10 (IL-10) expressions at 16 wpi were significantly lower in both female and male mice colonized by WT H. hepaticus or in males transiently colonized through 8 wpi by HhcdtBm7 versus control mice (P < 0.0159). These lines of evidence indicate that (i) H. hepaticus CDT plays a crucial role in the persistent colonization of H. hepaticus in SW mice; (ii) SW female mice are more resistant to H. hepaticus colonization than male mice; (iii) there was persistent colonization of WT H. hepaticus in cecum, colon, and jejunum but only transient colonization of H. hepaticus in the ileum of female mice; (iv) H. hepaticus colonization was associated with down-regulation of colonic IL-10 production.

Helicobacter hepaticus expresses a member of the cytolethal distending toxin (CDT) family of bacterial cytotoxins. To investigate the role of CDT in the pathogenesis of H. hepaticus, transposon mutagenesis was used to generate a series of isogenic mutants in and around the cdtABC gene cluster. An H. hepaticus transposon mutant with a disrupted cdtABC coding region no longer produced CDT activity. Conversely, a transposon insertion outside of the cluster did not affect the CDT activity. An examination of these mutants demonstrated that CDT represents the previously described granulating cytotoxin in H. hepaticus. Challenge of C57BL/6 interleukin 10(-/-) mice with isogenic H. hepaticus mutants revealed that CDT expression is not required for colonization of the murine gut. However, a CDT-negative H. hepaticus mutant had a significantly diminished capacity to induce lesions in this murine model of inflammatory bowel disease.

Gamma interferon-deficient (IFN-gamma KO) mice developed a wasting syndrome and were found to be co-infected with Helicobacter sp., and a new isolate of mouse hepatitis virus (MHV) designated MHV-G. The disease was characterized by pleuritis, peritonitis, hepatitis, pneumonia, and meningitis. Initial experiments used a cecal homogenate inoculum from the clinical cases that contained H. hepaticus and MHV-G to reproduce the development of peritonitis and pleuritis in IFN-gamma KO mice. In contrast, immunocompetent mice given the same inoculum developed an acute, self-limiting infection and remained clinically normal. This result confirmed the importance of IFN-gamma in preventing chronic infection and limiting viral dissemination. To understand the role of both agents in the development of peritonitis and pleuritis, IFN-gamma KO mice were infected with either agent or were co-infected with H. hepaticus and MHV-G. Infection with MHV-G induced a multisystemic infection similar to that described in the original cases, with multifocal hepatic necrosis, acute necrotizing and inflammatory lesions of the gastrointestinal tract, and acute peritonitis and pleuritis with adhesions on the serosal surfaces of the viscera. However, mice given H. hepaticus alone had minimal pathologic changes even though the organism was consistently detected in the cecum or feces. Although co-infection with H. hepaticus and MHV-G induced lesions similar to those associated with MHV-G alone, the pathogenesis of the MHV infection was modified. Helicobacter hepaticus appeared to reduce the severity of MHV-induced lesions during the acute phase of infection, and exacerbated hepatitis and meningitis at the later time point. We conclude that infection of IFN-gamma KO mice with MHV-G results in multisystemic infection with peritonitis, pleuritis, and adhesions due to the aberrant immune response in these mice. In addition, co-infection of these mice with H. hepaticus results in alterations in the pathogenesis of MHV-G infection.