Sustained exposure to Helicobacter pylori induces immune tolerance by desensitizing TLR6.

Helicobacter pylori (H. pylori, Hp) has been designated a class I carcinogen and is closely associated with severe gastric diseases. During colonization in the gastric mucosa, H. pylori develops immune escape by inducing host immune tolerance. The gastric epithelium acts as the first line of defense against H. pylori, with Toll-like receptors (TLRs) in gastric epithelial cells being sensitive to H. pylori components and subsequently activating the innate immune system. However, the mechanism of immune tolerance induced by H. pylori through the TLR signalling pathway has not been fully elucidated. In this research, we detected the expression of TLRs and inflammatory cytokines in GES-1 cells upon sustained exposure to H. pylori or H. pylori lysate from 1 to 30 generations and in Mongolian gerbils infected with H. pylori for 5 to 90 weeks. We found that the levels of TLR6 and inflammatory cytokines first increased and then dropped during the course of H. pylori treatment in vitro and in vivo. The restoration of TLR6 potentiated the expression of IL-1ß and IL-8 in GES-1 cells, which recruited neutrophils and reduced the colonization of H. pylori in the gastric mucosa of gerbils. Mechanistically, we found that persistent infection with H. pylori reduces the sensitivity of TLR6 to bacterial components and regulates the expression of inflammatory cytokines in GES-1 cells through TLR6/JNK signaling. The TLR6 agonist obviously alleviated inflammation in vitro and in vivo. Promising results suggest that TLR6 may be a potential candidate immunotherapy drug for H. pylori infection.

Convergent dysbiosis of gastric mucosa and fluid microbiome during stomach carcinogenesis.

BACKGROUND: A complex microbiota in the gastric mucosa (GM) has been unveiled recently and its dysbiosis is identified to be associated with gastric cancer (GC). However, the microbial composition in gastric fluid (GF) and its correlation with GM during gastric carcinogenesis are unclear. METHODS: We obtained GM and GF samples from 180 patients, including 61 superficial gastritis (SG), 55 intestinal metaplasia (IM) and 64 GC and performed 16S rRNA gene sequencing analysis. The concentration of gastric acid and metabolite nitrite has been measured. RESULTS: Overall, the composition of microbiome in GM was distinct from GF with less diversity, and both were influenced by H. pylori infection. The structure of microbiota changed differentially in GM and GF across histological stages of GC, accompanied with decreased gastric acid and increased carcinogenic nitrite. The classifiers of GC based on microbial markers were identified in both GM and GF, including Lactobacillus, Veillonella, Gemella, and were further validated in an independent cohort with good performance. Interestingly, paired comparison between GM and GF showed that their compositional distinction remarkably dwindled from SG to GC, with some GF-enriched bacteria significantly increased in GM. Moreover, stronger interaction network between microbes of GM and GF was observed in GC compared to SG. CONCLUSION: Our results, for the first time, revealed a comprehensive profile of both GM and GF microbiomes during the development of GC. The convergent microbial characteristics between GM and GF in GC suggest that the colonization of carcinogenic microbes in GM might derive from GF.

Gamma-glutamyltranspeptidase expression by Helicobacter saguini, an enterohepatic Helicobacter species isolated from cotton top tamarins with chronic colitis.

BACKGROUND: Helicobacter saguini is a novel enterohepatic Helicobacter species isolated from captive cotton top tamarins with chronic colitis and colon cancer. Monoassociated H. saguini infection in gnotobiotic IL-10-/- mice causes typhlocolitis and dysplasia; however, the virulent mechanisms of this species are unknown. Gamma-glutamyltranspeptidase (GGT) is an enzymatic virulence factor expressed by pathogenic Helicobacter and Campylobacter species that inhibits host cellular proliferation and promotes inflammatory-mediated gastrointestinal pathology. The aim of this study was to determine if H. saguini expresses an enzymatically active GGT homologue with virulence properties. EXPERIMENTAL PROCEDURES: Two putative GGT paralogs (HSGGT1 and HSGGT2) identified in the H. saguini genome were bioinformatically analysed to predict enzymatic functionality and virulence potential. An isogenic knockout mutant strain and purified recombinant protein of HSGGT1 were created to study enzymatic activity and virulence properties by in vitro biochemical and cell culture experiments. RESULTS: Bioinformatic analysis predicted that HSGGT1 has enzymatic functionality and is most similar to the virulent homologue expressed by Helicobacter bilis, whereas HSGGT2 contains putatively inactivating mutations. An isogenic knockout mutant strain and recombinant HSGGT1 protein were successfully created and demonstrated that H. saguini has GGT enzymatic activity. Recombinant HSGGT1 protein and sonicate from wild-type but not mutant H. saguini inhibited gastrointestinal epithelial and lymphocyte cell proliferation without evidence of cell death. The antiproliferative effect by H. saguini sonicate or recombinant HSGGT1 protein could be significantly prevented with glutamine supplementation or the GGT-selective inhibitor acivicin. Recombinant HSGGT1 protein also induced proinflammatory gene expression in colon epithelial cells. CONCLUSIONS: This study shows that H. saguini may express GGT as a potential virulence factor and supports further in vitro and in vitro studies into how GGT expression by enterohepatic Helicobacter species influences the pathogenesis of gastrointestinal inflammatory diseases.

Male-Dependent Promotion of Colitis in 129 Rag2-/- Mice Co-Infected with Helicobacter pylori and Helicobacter hepaticus.

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-1ß, 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.

Muc5ac null mice are predisposed to spontaneous gastric antro-pyloric hyperplasia and adenomas coupled with attenuated H. pylori-induced corpus mucous metaplasia.

Gastric cancer (GC) is the third leading cause of cancer-related deaths worldwide and is strongly associated with chronic Helicobacter pylori (Hp) infection. The ability of Hp to closely adhere to the gastric surface protective mucous layer containing mucins (MUC in humans and Muc in animals), primarily Muc5ac, is integral in the stepwise pathogenesis from gastritis to cancer. To probe the role of Muc5ac in Hp-induced gastric pathology, Muc5ac-/- and Muc5ac+/+ (WT) mice were experimentally infected with Hp Sydney strain (SS1). At 16 weeks and 32 weeks post infection (wpi), groups of mice were euthanized and evaluated for the following: gastric histopathological parameters, immunohistochemical expression of mucins (Muc5ac, Muc1, Muc2), Trefoil factor family proteins (Tff1 and Tff2), Griffonia (Bandeiraea) simplicifolia lectin II (GSL II) (mucous metaplasia marker) and Clusterin (Spasmolytic Polypeptide Expressing Metaplasia (SPEM) marker), Hp colonization density by qPCR and gastric cytokine mRNA levels. Our results demonstrate that Muc5ac-/- mice developed spontaneous antro-pyloric proliferation, adenomas and in one case with neuroendocrine differentiation; these findings were independent of Hp infection along with strong expression levels of Tff1, Tff2 and Muc1. Hp-infected Muc5ac-/- mice had significantly lowered gastric corpus mucous metaplasia at 16 wpi and 32 wpi (P = 0.0057 and P = 0.0016, respectively), with a slight reduction in overall gastric corpus pathology. GSII-positive mucous neck cells were decreased in Hp-infected Muc5ac-/- mice compared to WT mice and clusterin positivity was noted within metaplastic glands in both genotypes following Hp infection. Additionally, Hp colonization densities were significantly higher in Muc5ac-/- mice compared to WT at 16 wpi in both sexes (P = 0.05) along with a significant reduction in gastric Tnfα (16 wpi-males and females, P = 0.017 and P = 0.036, respectively and 32 wpi-males only, P = 0.025) and Il-17a (16 wpi-males) (P = 0.025). Taken together, our findings suggest a protective role for MUC5AC/Muc5ac in maintaining gastric antral equilibrium and inhibiting Hp colonization and associated inflammatory pathology.

Mutagenicity of Helicobacter hepaticus infection in the lower bowel mucosa of 129/SvEv Rag2-/- Il10-/- gpt delta mice is influenced by sex.

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.

Helicobacter hepaticus cytolethal distending toxin promotes intestinal carcinogenesis in 129Rag2-deficient mice.

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.

Male Syrian Hamsters Experimentally Infected with Helicobacter spp. of the H. bilis Cluster Develop MALT-Associated Gastrointestinal Lymphomas.

BACKGROUND: Aged hamsters naturally infected with novel Helicobacter spp. classified in the H. bilis cluster develop hepatobiliary lesions and typhlocolitis. METHODS: To determine whether enterohepatic H. spp. contribute to disease, Helicobacter-free hamsters were experimentally infected with H. spp. after suppression of intestinal bacteria by tetracycline treatment of dams and pups. After antibiotic withdrawal, weanlings were gavaged with four H. bilis-like Helicobacter spp. isolated from hamsters or H. bilis ATCC 43879 isolated from human feces and compared to controls (n = 7 per group). RESULTS: Helicobacter bilis 43879-dosed hamsters were necropsied at 33 weeks postinfection (WPI) due to the lack of detectable infection by fecal PCR; at necropsy, 5 of 7 were weakly PCR positive but lacked intestinal lesions. The remaining hamsters were maintained for ~95 WPI; chronic H. spp. infection in hamsters (6/7) was confirmed by PCR, bacterial culture, fluorescent in situ hybridization, and ELISA. Hamsters had mild-to-moderate typhlitis, and three of the male H. spp.-infected hamsters developed small intestinal lymphoma, in contrast to one control. Of the three lymphomas in H. spp.-infected hamsters, one was a focal ileal mucosa-associated lymphoid tissue (MALT) B-cell lymphoma, while the other two were multicentric small intestinal large B-cell lymphomas involving both the MALT and extra-MALT mucosal sites with lymphoepithelial lesions. The lymphoma in the control hamster was a diffuse small intestinal lymphoma with a mixed population of T and B cells. CONCLUSIONS: Results suggest persistent H. spp. infection may augment risk for gastrointestinal MALT origin lymphomas. This model is consistent with H. pylori/heilmannii-associated MALT lymphoma in humans and could be further utilized to investigate the mechanisms of intestinal lymphoma development.

Infection-induced colitis in mice causes dynamic and tissue-specific changes in stress response and DNA damage leading to colon cancer.

Helicobacter hepaticus-infected Rag2(-/-) mice emulate many aspects of human inflammatory bowel disease, including the development of colitis and colon cancer. To elucidate mechanisms of inflammation-induced carcinogenesis, we undertook a comprehensive analysis of histopathology, molecular damage, and gene expression changes during disease progression in these mice. Infected mice developed severe colitis and hepatitis by 10 wk post-infection, progressing into colon carcinoma by 20 wk post-infection, with pronounced pathology in the cecum and proximal colon marked by infiltration of neutrophils and macrophages. Transcriptional profiling revealed decreased expression of DNA repair and oxidative stress response genes in colon, but not in liver. Mass spectrometric analysis revealed higher levels of DNA and RNA damage products in liver compared to colon and infection-induced increases in 5-chlorocytosine in DNA and RNA and hypoxanthine in DNA. Paradoxically, infection was associated with decreased levels of DNA etheno adducts. Levels of nucleic acid damage from the same chemical class were strongly correlated in both liver and colon. The results support a model of inflammation-mediated carcinogenesis involving infiltration of phagocytes and generation of reactive species that cause local molecular damage leading to cell dysfunction, mutation, and cell death. There are strong correlations among histopathology, phagocyte infiltration, and damage chemistry that suggest a major role for neutrophils in inflammation-associated cancer progression. Further, paradoxical changes in nucleic acid damage were observed in tissue- and chemistry-specific patterns. The results also reveal features of cell stress response that point to microbial pathophysiology and mechanisms of cell senescence as important mechanistic links to cancer.

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-ß, 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.

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.

Hypoxia exacerbates the malignant transformation of gastric epithelial cells induced by long-term H. pylori infection.

Helicobacter pylori is a microaerophilic Gram-negative bacterium that resides in the human stomach and is classified as a class I carcinogen for gastric cancer. Numerous studies have demonstrated that H. pylori infection plays a role in regulating the function of host cells, thereby contributing to the malignant transformation of these cells. However, H. pylori infection is a chronic process, and short-term cellular experiments may not provide a comprehensive understanding of the in vivo situation, especially when considering the lower oxygen levels in the human stomach. In this study, we aimed to investigate the mechanisms underlying gastric cell dysfunction after prolonged exposure to H. pylori under hypoxic conditions. We conducted a co-culture experiment using the gastric cell line GES-1 and H. pylori for 30 generations under intermittent hypoxic conditions. By closely monitoring cell proliferation, migration, invasion, autophagy, and apoptosis, we revealed that sustained H. pylori stimulation under hypoxic conditions significantly influences the function of GES-1 cells. This stimulation induces epithelial-mesenchymal transition and contributes to the propensity for malignant transformation of gastric cells. To confirm the in vitro results, we conducted an experiment involving Mongolian gerbils infected with H. pylori for 85 weeks. All the results strongly suggest that the Nod1 receptor signaling pathway plays a crucial role in H. pylori-related apoptosis and autophagy. In summary, continuous stimulation by H. pylori affects the functioning of gastric cells through the Nod1 receptor signaling pathway, increasing the likelihood of cell carcinogenesis. The presence of hypoxic conditions further exacerbates this process.IMPORTANCEDeciphering the collaborative effects of Helicobacter pylori infection on gastric epithelial cell function is key to unraveling the development mechanisms of gastric cancer. Prior research has solely examined the outcomes of short-term H. pylori stimulation on gastric epithelial cells under aerobic conditions, neglecting the bacterium's nature as a microaerophilic organism that leads to cancer following prolonged stomach colonization. This study mimics a more genuine in vivo infection scenario by repeatedly exposing gastric epithelial cells to H. pylori under hypoxic conditions for up to 30 generations. The results show that chronic exposure to H. pylori in hypoxia substantially increases cell migration, invasion, and epithelial-mesenchymal transition, while suppressing autophagy and apoptosis. This highlights the significance of hypoxic conditions in intensifying the carcinogenic impact of H. pylori infection. By accurately replicating the in vivo gastric environment, this study enhances our comprehension of H. pylori's pathogenic mechanisms in gastric cancer.

IL-1ß transgenic mouse model of inflammation driven esophageal and oral squamous cell carcinoma.

Chronic inflammation is integral to the development of esophageal adenocarcinoma (EAC) and esophageal squamous cell carcinoma (ESCC), although the latter has not been associated with reflux esophagitis. The L2-IL-1ß transgenic mice, expressing human interleukin (IL)-1ß in the oral, esophageal and forestomach squamous epithelia feature chronic inflammation and a stepwise development of Barrett's esophagus-like metaplasia, dysplasia and adenocarcinoma at the squamo-columnar junction. However, the functional consequences of IL-1ß-mediated chronic inflammation in the oral and esophageal squamous epithelia remain elusive. We report for the first time that in addition to the previously described Barrett's esophagus-like metaplasia, the L2-IL-1ß mice also develop squamous epithelial dysplasia with progression to squamous cell carcinoma (SCC) in the esophagus and the tongue. L2-IL-1ß showed age-dependent progression of squamous dysplasia to SCC with approximately 40% (n = 49) and 23.5% (n = 17) incidence rates for esophageal and tongue invasive SCC respectively, by 12-15 months of age. Interestingly, SCC development and progression in L2-IL-1ß was similar in both Germ Free (GF) and Specific Pathogen Free (SPF) conditions. Immunohistochemistry revealed a T cell predominant inflammatory profile with enhanced expression of Ki67, Sox2 and the DNA double-strand break marker, γ-H2AX, in the dysplastic squamous epithelia of L2-IL-1ß mice. Pro-inflammatory cytokines, immunomodulatory players, chemoattractants for inflammatory cells (T cells, neutrophils, eosinophils, and macrophages) and oxidative damage marker, iNOS, were significantly increased in the esophageal and tongue tissues of L2-IL-1ß mice. Our recent findings have expanded the translational utility of the IL-1ß mouse model to aid in further characterization of the key pathways of inflammation driven BE and EAC as well as ESCC and Oral SCC.

Effects of chronic Helicobacter pylori strain PMSS1 infection on whole brain and gastric iron homeostasis in male INS-GAS mice.

Iron deficiency, the most common micronutrient deficiency in humans, is associated with long-term deficits in cognition and memory if left untreated. Infection with the gastric pathogen Helicobacter pylori has been linked to iron deficiency anemia (IDA). The H. pylori virulence factor cytotoxin-associated gene A (cagA) is proposed to be especially pertinent in iron deficiency. Male INS-GAS/FVB mice were infected with the CagA+ strain pre-murine Sydney strain 1 (PMSS1) for 12-13 or 27-29 weeks to investigate the role of chronic H. pylori infection in iron deficiency and neurological sequelae. Mice at both timepoints demonstrated significantly elevated gastric histopathology scores and inflammatory cytokines compared to sham-dosed controls. However, only mice at 27-29 weeks post infection had changes in hematological parameters, with significantly decreased erythrocyte count, hematocrit, serum hemoglobin, and increased serum total iron binding capacity. Gastric transcription of iron-regulatory genes Hamp and Bmp4 were significantly downregulated at both timepoints. In the brain, iron-dependent myelingergic and synaptic markers were significantly downregulated at 27-29 weeks. These results indicated that long-term infection of the CagA + PMSS1 strain of H. pylori in this study caused anemia, altered gastric iron homeostasis, and neurological changes similar to those reported in other rodent H. pylori CagA- strain infection models.

YAP and ß-catenin cooperate to drive H. pylori-induced gastric tumorigenesis.

H. pylori infection is the strongest known risk factor for gastric carcinoma. The activation of the yes-associated protein 1 (YAP) and ß-catenin pathways has been associated with multiple tumor types. In this study, we investigated the crosstalk between the YAP and ß-catenin pathways in H. pylori-associated gastric tumorigenesis. Immunohistochemical analysis of YAP and ß-catenin expression was performed in human gastric cancer tissues. The small molecules Super-TDU and KYA1797K, pharmacological inhibitors of YAP and ß-catenin, respectively, were used to investigate the role of these signaling pathways in H. pylori-induced gastric carcinogenesis in murine models of infection. The common downstream targets of YAP and ß-catenin signaling were evaluated by RNA sequencing (RNA-seq). Western blot, immunofluorescence, luciferase, RT-PCR, immunoprecipitation, cell counting kit-8 (CCK8), EdU and spheroid assays were used. H. pylori infection promoted YAP and ß-catenin nuclear accumulation and transcriptional activity in gastric epithelial cells and transgenic insulin-gastrin (INS-GAS) mice, whereas silencing of both YAP and ß-catenin synergistically inhibited H. pylori-induced cell proliferation and expansion. In addition, YAP was found to directly interact with ß-catenin and knockdown of YAP suppressed H. pylori-induced nuclear translocation of ß-catenin. Moreover, downstream genes caudal-type homeobox 2 (CDX2), leucine-rich repeat containing G protein-coupled receptor 5 (LGR5) and RuvB like AAA ATPase 1 (RUVBL1) were shared by both YAP and ß-catenin signaling. Furthermore, treatment with the YAP inhibitor Super-TDU or ß-catenin inhibitor KYA1797A significantly alleviated gastric inflammation and epithelial DNA damage in H. pylori-infected mice. Finally, the elevation of gastric YAP was positively correlated with ß-catenin expression in human gastric cancer tissues. These findings indicate that YAP and ß-catenin synergistically promote H. pylori-induced gastric carcinogenesis via their physical interaction and reveal that CDX2, LGR5 and RUVBL1 are the downstream genes shared by both the YAP and ß-catenin signaling pathways, and potentially contribute to H. pylori pathogenesis.

Systemic macrophage depletion inhibits Helicobacter bilis-induced proinflammatory cytokine-mediated typhlocolitis and impairs bacterial colonization dynamics in a BALB/c Rag2-/- mouse model of inflammatory bowel disease.

Helicobacter bilis, an enterohepatic helicobacter, is associated with chronic hepatitis in aged immunocompetent inbred mice and inflammatory bowel disease (IBD) in immunodeficient mice. To evaluate the role of macrophages in H. bilis-induced IBD, Rag2(-/-) BALB/c or wild-type (WT) BALB/c mice were either sham dosed or infected with H. bilis Missouri strain under specific-pathogen-free conditions, followed by an intravenous injection of a 0.2-ml suspension of liposomes coated with either phosphate-buffered saline (control) or clodronate (a macrophage depleting drug) at 15 weeks postinfection (wpi). At 16 wpi, the ceca of H. bilis-infected Rag2(-/-) mice treated with control liposomes had significantly higher histopathological lesional scores (for cumulative typhlitis index, inflammation, edema, epithelial defects, and hyperplasia) and higher counts of F4/80(+) macrophages and MPO(+) neutrophils compared to H. bilis-infected Rag2(-/-) mice treated with clodronate liposomes. In addition, cecal quantitative PCR analyses revealed a significant suppression in the expression of macrophage-related cytokine genes, namely, Tnfa, Il-1ß, Il-10, Cxcl1, and iNos, in the clodronate-treated H. bilis-infected Rag2(-/-) mice compared to the H. bilis-infected Rag2(-/-) control mice. Finally, cecal quantitative PCR analyses also revealed a significant reduction in bacterial colonization in the clodronate-treated Rag2(-/-) mice. Taken together, our results suggest that macrophages are critical inflammatory cellular mediators for promoting H. bilis-induced typhlocolitis in mice.

Lack of commensal flora in Helicobacter pylori-infected INS-GAS mice reduces gastritis and delays intraepithelial neoplasia.

BACKGROUND & AIMS: Transgenic FVB/N insulin-gastrin (INS-GAS) mice have high circulating gastrin levels, and develop spontaneous atrophic gastritis and gastrointestinal intraepithelial neoplasia (GIN) with 80% prevalence 6 months after Helicobacter pylori infection. GIN is associated with gastric atrophy and achlorhydria, predisposing mice to nonhelicobacter microbiota overgrowth. We determined if germfree INS-GAS mice spontaneously develop GIN and if H pylori accelerates GIN in gnotobiotic INS-GAS mice. METHODS: We compared gastric lesions, levels of messenger RNA, serum inflammatory mediators, antibodies, and gastrin among germfree and H pylori-monoinfected INS-GAS mice. Microbiota composition of specific pathogen-free (SPF) INS-GAS mice was quantified by pyrosequencing. RESULTS: Germfree INS-GAS mice had mild hypergastrinemia but did not develop significant gastric lesions until 9 months old and did not develop GIN through 13 months. H pylori monoassociation caused progressive gastritis, epithelial defects, oxyntic atrophy, marked foveolar hyperplasia, dysplasia, and robust serum and tissue proinflammatory immune responses (particularly males) between 5 and 11 months postinfection (P<0.05, compared with germfree controls). Only 2 of 26 female, whereas 8 of 18 male, H pylori-infected INS-GAS mice developed low to high-grade GIN by 11 months postinfection. Stomachs of H pylori-infected SPF male mice had significant reductions in Bacteroidetes and significant increases in Firmicutes. CONCLUSIONS: Gastric lesions take 13 months longer to develop in germfree INS-GAS mice than male SPF INS-GAS mice. H pylori monoassociation accelerated gastritis and GIN but caused less severe gastric lesions and delayed onset of GIN compared with H pylori-infected INS-GAS mice with complex gastric microbiota. Changes in gastric microbiota composition might promote GIN in achlorhydric stomachs of SPF mice.

The Epidemiology of Invasive, Multipleantibiotic-resistant Klebsiella pneumoniae Infection in a Breeding Colony of Immunocompromised NSG Mice.

Klebsiella pneumoniae (Kp) is a gram-negative opportunistic pathogen that causes severe pneumonia, pyelonephritis, and sepsis in immunocompromised hosts. During a 4-mo interval, several NOD.Cg-PrkdcscidIl2rgtm1Wjl/SzJ (NSG) breeders and pups in our facilities were diagnosed with Kp infections. An initial 6 adult and 1 juvenile NSG mice were submitted for necropsy and histologic examination because of acute onset of diarrhea and death. The evaluation revealed typhlocolitis in 2 of the mice and tritrichomoniasis in all 7. Escherichia coli positive for polyketide synthase (pks+) and Kp were isolated from the intestines. Given a history of sepsis due to pks+ E. coli in NSG mice in our facilities and determination of its antimicrobial susceptibility, trimethoprim-sulfamethoxazole (TMP-SMX) was administered to the colony in the drinking water for 4 wk. After this intervention, an additional 21 mice became ill or died; 11 of these mice had suppurative pneumonia, meningoencephalitis, hepatitis, metritis, pyelonephritis, or sepsis. Kp was cultured from pulmonary abscesses or blood of 10 of the mice. Whole-genome sequencing (WGS) indicated that the Kp isolates contained genes associated with phenotypes found in pore-forming Kp isolates cultured from humans with ulcerative colitis and primary sclerosing cholangitis. None of the Kp isolates exhibited a hyperviscous phenotype, but 13 of 14 were resistant to TMP-SMX. Antimicrobial susceptibility testing indicated sensitivity of the Kp to enrofloxacin, which was administered in the drinking water. Antibiotic sensitivity profiles were confirmed by WGS of the Kp strains; key virulence and resistance genes to quaternary ammonia compounds were also identified. Enrofloxacin treatment resulted in a marked reduction in mortality, and the study using the NSG mice was completed successfully. Our findings implicate intestinal translocation of Kp as the cause of pneumonia and systemic infections in NSG mice and highlight the importance of identification of enteric microbial pathogens and targeted antibiotic selection when treating bacterial infections in immunocompromised mice.

A positive feedback loop of the TAZ/ß-catenin axis promotes Helicobacter pylori-associated gastric carcinogenesis.

Background: Helicobacter pylori infection is the strongest known risk factor for gastric cancer. The Hippo signaling pathway controls organ size and maintains tissue homeostasis by coordinately regulating cell growth and proliferation. Here, we demonstrate the interactive role of TAZ, the transcriptional coactivator of the Hippo pathway, and beta-catenin in promoting the pathogenesis of H. pylori infection. Methods: TAZ expression was evaluated in human gastric tissues and H. pylori-infected insulin-gastrin (INS-GAS) mice. Western blot, immunofluorescence, immunohistochemistry, and RT-PCR assays were performed. Coimmunoprecipitation was performed to examine the interaction between TAZ and ß-catenin. TAZ and ß-catenin were silenced using small interfering RNAs. HA-ß-catenin and Flag-TAZ were constructed. Results: Increased TAZ was noted in human gastric cancer tissues compared to chronic gastritis tissues and in H. pylori-positive gastritis tissues compared to H. pylori-negative gastritis tissues. In addition, H. pylori infection induced TAZ expression and nuclear accumulation in the gastric tissue of INS-GAS mice and cultured gastric epithelial cells, which was dependent on the virulence factor CagA. Moreover, TAZ or ß-catenin knockdown significantly suppressed H. pylori infection-induced cell growth, survival, and invasion. Furthermore, the interactive regulation of TAZ and ß-catenin activation was revealed. Finally, ß-catenin was required for H. pylori-induced TAZ activation. Conclusion: These findings suggest the existence of a positive feedback loop of activation between TAZ and ß-catenin that could play an important role in CagA+ H. pylori infection-induced gastric carcinogenesis. TAZ inhibition represents a potential target for the prevention of H. pylori infection-associated gastric cancer.

17 ß-estradiol suppresses Helicobacter pylori-induced gastric pathology in male hypergastrinemic INS-GAS mice.

Helicobacter pylori-associated gastric cancer is male predominant and animal studies suggest that sex hormones influence gastric carcinogenesis. We investigated the effects of 17ß-estradiol (E2) or castration on H.pylori-induced gastritis in male INS-GAS/FVB/N (Tg(Ins1-GAS)1Sbr) mice. Comparisons were made to previously evaluated sham (n = 8) and H.pylori-infected (n = 8), intact male INS-GAS mice which had developed severe corpus gastritis accompanied by atrophy, hyperplasia, intestinal metaplasia and dysplasia of the epithelium within 16 weeks postinfection (all P < 0.01). Castration at 8 weeks of age had no sparing effect on lesions in uninfected (n = 5) or H.pylori-infected mice (n = 7) but all lesion subfeatures were attenuated by E2 in H.pylori-infected mice (n = 7) (P < 0.001). Notably, inflammation was not reduced but glandular atrophy, hyperplasia, intestinal metaplasia and dysplasia were also less severe in uninfected, E2-treated mice (n = 7) (P < 0.01). Attenuation of gastric lesions by E2 was associated with lower messenger RNA (mRNA) expression of interferon (IFN)-γ (P < 0.05) and interleukin (IL)-1ß (P < 0.004), and higher IL-10 (P < 0.02) as well as decreased numbers of Foxp3(+) regulatory T cells when compared with infected intact males. Infected E2-treated mice also developed higher Th2-associated anti-H.pylori IgG1 responses (P < 0.05) and significantly lower Ki-67 indices of epithelial proliferation (P < 0.05). E2 elevated expression of mRNA for Foxp3 (P < 0.0001) and IL-10 (P < 0.01), and decreased IL-1ß (P < 0.01) in uninfected, intact male mice compared with controls. Therefore, estrogen supplementation, but not castration, attenuated gastric lesions in H.pylori-infected male INS-GAS mice and to a lesser extent in uninfected mice, potentially by enhancing IL-10 function, which in turn decreased IFN-γ and IL-1ß responses induced by H.pylori.