Hypusination Maintains Intestinal Homeostasis and Prevents Colitis and Carcinogenesis by Enhancing Aldehyde Detoxification.

BACKGROUND & AIMS: The amino acid hypusine, synthesized from the polyamine spermidine by the enzyme deoxyhypusine synthase (DHPS), is essential for the activity of eukaryotic translation initiation factor 5A (EIF5A). The role of hypusinated EIF5A (EIF5AHyp) remains unknown in intestinal homeostasis. Our aim was to investigate EIF5AHyp in the gut epithelium in inflammation and carcinogenesis. METHODS: We used human colon tissue messenger RNA samples and publicly available transcriptomic datasets, tissue microarrays, and patient-derived colon organoids. Mice with intestinal epithelial-specific deletion of Dhps were investigated at baseline and in models of colitis and colon carcinogenesis. RESULTS: We found that patients with ulcerative colitis and Crohn's disease exhibit reduced colon levels of DHPS messenger RNA and DHPS protein and reduced levels of EIF5AHyp. Similarly, colonic organoids from colitis patients also show down-regulated DHPS expression. Mice with intestinal epithelial-specific deletion of Dhps develop spontaneous colon hyperplasia, epithelial proliferation, crypt distortion, and inflammation. Furthermore, these mice are highly susceptible to experimental colitis and show exacerbated colon tumorigenesis when treated with a carcinogen. Transcriptomic and proteomic analysis on colonic epithelial cells demonstrated that loss of hypusination induces multiple pathways related to cancer and immune response. Moreover, we found that hypusination enhances translation of numerous enzymes involved in aldehyde detoxification, including glutathione S-transferases and aldehyde dehydrogenases. Accordingly, hypusination-deficient mice exhibit increased levels of aldehyde adducts in the colon, and their treatment with a scavenger of electrophiles reduces colitis. CONCLUSIONS: Hypusination in intestinal epithelial cells has a key role in the prevention of colitis and colorectal cancer, and enhancement of this pathway via supplementation of spermidine could have a therapeutic impact.

Ornithine Decarboxylase in Gastric Epithelial Cells Promotes the Immunopathogenesis of Helicobacter pylori Infection.

Colonization by Helicobacter pylori is associated with gastric diseases, ranging from superficial gastritis to more severe pathologies, including intestinal metaplasia and adenocarcinoma. The interplay of the host response and the pathogen affect the outcome of disease. One major component of the mucosal response to H. pylori is the activation of a strong but inefficient immune response that fails to control the infection and frequently causes tissue damage. We have shown that polyamines can regulate H. pylori-induced inflammation. Chemical inhibition of ornithine decarboxylase (ODC), which generates the polyamine putrescine from l-ornithine, reduces gastritis in mice and adenocarcinoma incidence in gerbils infected with H. pylori However, we have also demonstrated that Odc deletion in myeloid cells enhances M1 macrophage activation and gastritis. Here we used a genetic approach to assess the specific role of gastric epithelial ODC during H. pylori infection. Specific deletion of the gene encoding for ODC in gastric epithelial cells reduces gastritis, attenuates epithelial proliferation, alters the metabolome, and downregulates the expression of immune mediators induced by H. pylori Inhibition of ODC activity or ODC knockdown in human gastric epithelial cells dampens H. pylori-induced NF-κB activation, CXCL8 mRNA expression, and IL-8 production. Chronic inflammation is a major risk factor for the progression to more severe pathologies associated with H. pylori infection, and we now show that epithelial ODC plays an important role in mediating this inflammatory response.

Protective Role of Spermidine in Colitis and Colon Carcinogenesis.

BACKGROUND & AIMS: Because inflammatory bowel disease is increasing worldwide and can lead to colitis-associated carcinoma (CAC), new interventions are needed. We have shown that spermine oxidase (SMOX), which generates spermidine (Spd), regulates colitis. Here we determined whether Spd treatment reduces colitis and carcinogenesis. METHODS: SMOX was quantified in human colitis and associated dysplasia using quantitative reverse-transcription polymerase chain reaction and immunohistochemistry. We used wild-type (WT) and Smox-/- C57BL/6 mice treated with dextran sulfate sodium (DSS) or azoxymethane (AOM)-DSS as models of colitis and CAC, respectively. Mice with epithelial-specific deletion of Apc were used as a model of sporadic colon cancer. Animals were supplemented or not with Spd in the drinking water. Colonic polyamines, inflammation, tumorigenesis, transcriptomes, and microbiomes were assessed. RESULTS: SMOX messenger RNA levels were decreased in human ulcerative colitis tissues and inversely correlated with disease activity, and SMOX protein was reduced in colitis-associated dysplasia. DSS colitis and AOM-DSS-induced dysplasia and tumorigenesis were worsened in Smox-/- vs WT mice and improved in both genotypes with Spd. Tumor development caused by Apc deletion was also reduced by Spd. Smox deletion and AOM-DSS treatment were both strongly associated with increased expression of α-defensins, which was reduced by Spd. A shift in the microbiome, with reduced abundance of Prevotella and increased Proteobacteria and Deferribacteres, occurred in Smox-/- mice and was reversed with Spd. CONCLUSIONS: Loss of SMOX is associated with exacerbated colitis and CAC, increased α-defensin expression, and dysbiosis of the microbiome. Spd supplementation reverses these phenotypes, indicating that it has potential as an adjunctive treatment for colitis and chemopreventive for colon carcinogenesis.

The Colombian Chemoprevention Trial: 20-Year Follow-Up of a Cohort of Patients With Gastric Precancerous Lesions.

BACKGROUND & AIMS: Helicobacter pylori eradication and endoscopic surveillance of gastric precancerous lesions are strategies to reduce gastric cancer (GC) risk. To our knowledge, this study is the longest prospective cohort of an H pylori eradication trial in a Hispanic population. METHODS: A total of 800 adults with precancerous lesions were randomized to anti-H pylori treatment or placebo. Gastric biopsy samples taken at baseline and 3, 6, 12, 16, and 20 years were assessed by our Correa histopathology score. A generalized linear mixed model with a participant-level random intercept was used to estimate the effect of H pylori status on the score over time. Logistic regression models were used to estimate progression by baseline diagnosis and to estimate GC risk by intestinal metaplasia (IM) subtype and anatomic location. RESULTS: Overall, 356 individuals completed 20 years of follow-up. Anti-H pylori therapy (intention-to-treat) reduced progression of the Correa score (odds ratio [OR], 0.59; 95% confidence interval [CI], 0.38-0.93). H pylori-negative status had a beneficial effect on the score over time (P = .036). Among individuals with IM (including indefinite for dysplasia) at baseline, incidence rates per 100 person-years were 1.09 (95% CI, 0.85-1.33) for low-grade/high-grade dysplasia and 0.14 (95% CI, 0.06-0.22) for GC. Incomplete-type (vs complete-type) IM at baseline presented higher GC risk (OR, 13.4; 95% CI, 1.8-103.8). Individuals with corpus (vs antrum-restricted) IM showed an OR of 2.1 (95% CI, 0.7-6.6) for GC. CONCLUSIONS: In a high-GC-risk Hispanic population, anti-H pylori therapy had a long-term beneficial effect against histologic progression. Incomplete IM is a strong predictor of GC risk.

Dicarbonyl Electrophiles Mediate Inflammation-Induced Gastrointestinal Carcinogenesis.

BACKGROUND & AIMS: Inflammation in the gastrointestinal tract may lead to the development of cancer. Dicarbonyl electrophiles, such as isolevuglandins (isoLGs), are generated from lipid peroxidation during the inflammatory response and form covalent adducts with amine-containing macromolecules. Thus, we sought to determine the role of dicarbonyl electrophiles in inflammation-associated carcinogenesis. METHODS: The formation of isoLG adducts was analyzed in the gastric tissues of patients infected with Helicobacter pylori from gastritis to precancerous intestinal metaplasia, in human gastric organoids, and in patients with colitis and colitis-associated carcinoma (CAC). The effect on cancer development of a potent scavenger of dicarbonyl electrophiles, 5-ethyl-2-hydroxybenzylamine (EtHOBA), was determined in transgenic FVB/N insulin-gastrin (INS-GAS) mice and Mongolian gerbils as models of H pylori-induced carcinogenesis and in C57BL/6 mice treated with azoxymethane-dextran sulfate sodium as a model of CAC. The effect of EtHOBA on mutations in gastric epithelial cells of H pylori-infected INS-GAS mice was assessed by whole-exome sequencing. RESULTS: We show increased isoLG adducts in gastric epithelial cell nuclei in patients with gastritis and intestinal metaplasia and in human gastric organoids infected with H pylori. EtHOBA inhibited gastric carcinoma in infected INS-GAS mice and gerbils and attenuated isoLG adducts, DNA damage, and somatic mutation frequency. Additionally, isoLG adducts were elevated in tissues from patients with colitis, colitis-associated dysplasia, and CAC as well as in dysplastic tumors of C57BL/6 mice treated with azoxymethane-dextran sulfate sodium. In this model, EtHOBA significantly reduced adduct formation, tumorigenesis, and dysplasia severity. CONCLUSIONS: Dicarbonyl electrophiles represent a link between inflammation and somatic genomic alterations and are thus key targets for cancer chemoprevention.

Targeted mobilization of Lrig1+ gastric epithelial stem cell populations by a carcinogenic Helicobacter pylori type IV secretion system.

Helicobacter pylori-induced gastritis is the strongest risk factor for gastric adenocarcinoma, a malignancy preceded by a series of well-defined histological stages, including metaplasia. One microbial constituent that augments cancer risk is the cag type 4 secretion system (T4SS), which translocates the oncoprotein CagA into host cells. Aberrant stem cell activation is linked to carcinogenesis, and Lrig1 (leucine-rich repeats and Ig-like domains 1) marks a distinct population of progenitor cells. We investigated whether microbial effectors with carcinogenic potential influence Lrig1 progenitor cells ex vivo and via lineage expansion within H. pylori-infected gastric mucosa. Lineage tracing was induced in Lrig1-CreERT2/+;R26R-YFP/+ (Lrig1/YFP) mice that were uninfected or subsequently infected with cag+H. pylori or an isogenic cagE- mutant (nonfunctional T4SS). In contrast to infection with wild-type (WT) H. pylori for 2 wk, infection for 8 wk resulted in significantly increased inflammation and proliferation in the corpus and antrum compared with uninfected or mice infected with the cagE- mutant. WT H. pylori-infected mice harbored significantly higher numbers of Lrig1/YFP epithelial cells that coexpressed UEA1 (surface cell marker). The number of cells coexpressing intrinsic factor (chief cell marker), YFP (lineage marker), and GSII lectin (spasmolytic polypeptide-expressing metaplasia marker) were increased only by WT H. pylori In human samples, Lrig1 expression was significantly increased in lesions with premalignant potential compared with normal mucosa or nonatrophic gastritis. In conclusion, chronic H. pylori infection stimulates Lrig1-expressing progenitor cells in a cag-dependent manner, and these reprogrammed cells give rise to a full spectrum of differentiated cells.

The Innate Immune Glycoprotein Lactoferrin Represses the Helicobacter pylori cag Type IV Secretion System.

Chronic infection with Helicobacter pylori increases risk of gastric diseases including gastric cancer. Despite development of a robust immune response, H. pylori persists in the gastric niche. Progression of gastric inflammation to serious disease outcomes is associated with infection with H. pylori strains which encode the cag Type IV Secretion System (cag T4SS). The cag T4SS is responsible for translocating the oncogenic protein CagA into host cells and inducing pro-inflammatory and carcinogenic signaling cascades. Our previous work demonstrated that nutrient iron modulates the activity of the T4SS and biogenesis of T4SS pili. In response to H. pylori infection, the host produces a variety of antimicrobial molecules, including the iron-binding glycoprotein, lactoferrin. Our work shows that apo-lactoferrin exerts antimicrobial activity against H. pylori under iron-limited conditions, while holo-lactoferrin enhances bacterial growth. Culturing H. pylori in the presence of holo-lactoferrin prior to co-culture with gastric epithelial cells, results in repression of the cag T4SS activity. Concomitantly, a decrease in biogenesis of cag T4SS pili at the host-pathogen interface was observed under these culture conditions by high-resolution electron microscopy analyses. Taken together, these results indicate that acquisition of alternate sources of nutrient iron plays a role in regulating the pro-inflammatory activity of a bacterial secretion system and present novel therapeutic targets for the treatment of H. pylori-related disease.

Carcinogenic Helicobacter pylori Strains Selectively Dysregulate the In Vivo Gastric Proteome, Which May Be Associated with Stomach Cancer Progression.

Helicobacter pylori is the strongest risk factor for gastric cancer. Initial interactions between H. pylori and its host originate at the microbial-gastric epithelial cell interface, and contact between H. pylori and gastric epithelium activates signaling pathways that drive oncogenesis. One microbial constituent that increases gastric cancer risk is the cag pathogenicity island, which encodes a type IV secretion system that translocates the effector protein, CagA, into host cells. We previously demonstrated that infection of Mongolian gerbils with a carcinogenic cag+H. pylori strain, 7.13, recapitulates many features of H. pylori-induced gastric cancer in humans. Therefore, we sought to define gastric proteomic changes induced by H. pylori that are critical for initiation of the gastric carcinogenic cascade. Gastric cell scrapings were harvested from H. pylori-infected and uninfected gerbils for quantitative proteomic analyses using isobaric tags for relative and absolute quantitation (iTRAQ). Quantitative proteomic analysis of samples from two biological replicate experiments quantified a total of 2764 proteins, 166 of which were significantly altered in abundance by H. pylori infection. Pathway mapping identified significantly altered inflammatory and cancer-signaling pathways that included Rab/Ras signaling proteins. Consistent with the iTRAQ results, RABEP2 and G3BP2 were significantly up-regulated in vitro, ex vivo in primary human gastric monolayers, and in vivo in gerbil gastric epithelium following infection with H. pylori strain 7.13 in a cag-dependent manner. Within human stomachs, RABEP2 and G3BP2 expression in gastric epithelium increased in parallel with the severity of premalignant and malignant lesions and was significantly elevated in intestinal metaplasia and dysplasia, as well as gastric adenocarcinoma, compared with gastritis alone. These results indicate that carcinogenic strains of H. pylori induce dramatic and specific changes within the gastric proteome in vivo and that a subset of altered proteins within pathways with oncogenic potential may facilitate the progression of gastric carcinogenesis in humans.

Epidermal growth factor receptor inhibition downregulates Helicobacter pylori-induced epithelial inflammatory responses, DNA damage and gastric carcinogenesis.

OBJECTIVE: Gastric cancer is the third leading cause of cancer death worldwide and infection by Helicobacter pylori is the strongest risk factor. We have reported increased epidermal growth factor receptor (EGFR) phosphorylation in the H. pylori-induced human carcinogenesis cascade, and association with DNA damage. Our goal was to determine the role of EGFR activation in gastric carcinogenesis. DESIGN: We evaluated gefitinib, a specific EGFR inhibitor, in chemoprevention of H. pylori-induced gastric inflammation and cancer development. Mice with genetically targeted epithelial cell-specific deletion of Egfr (EfgrΔepi mice) were also used. RESULTS: In C57BL/6 mice, gefitinib decreased Cxcl1 and Cxcl2 expression by gastric epithelial cells, myeloperoxidase-positive inflammatory cells in the mucosa and epithelial DNA damage induced by H. pylori infection. Similar reductions in chemokines, inflammatory cells and DNA damage occurred in infected EgfrΔepi versus Egfrfl/fl control mice. In H. pylori-infected transgenic insulin-gastrin (INS-GAS) mice and gerbils, gefitinib treatment markedly reduced dysplasia and carcinoma. Gefitinib blocked H. pylori-induced activation of mitogen-activated protein kinase 1/3 (MAPK1/3) and activator protein 1 in gastric epithelial cells, resulting in inhibition of chemokine synthesis. MAPK1/3 phosphorylation and JUN activation was reduced in gastric tissues from infected wild-type and INS-GAS mice treated with gefitinib and in primary epithelial cells from EfgrΔepi versus Egfrfl/fl mice. Epithelial EGFR activation persisted in humans and mice after H. pylori eradication, and gefitinib reduced gastric carcinoma in INS-GAS mice treated with antibiotics. CONCLUSIONS: These findings suggest that epithelial EGFR inhibition represents a potential strategy to prevent development of gastric carcinoma in H. pylori-infected individuals.

Dynamics of Helicobacter pylori infection as a determinant of progression of gastric precancerous lesions: 16-year follow-up of an eradication trial.

OBJECTIVE: To evaluate the long-term effect of cumulative time exposed to Helicobacter pylori infection on the progression of gastric lesions. DESIGN: 795 adults with precancerous gastric lesions were randomised to receive anti-H. pylori treatment at baseline. Gastric biopsies were obtained at baseline and at 3, 6, 12 and 16 years. A total of 456 individuals attended the 16-year visit. Cumulative time of H. pylori exposure was calculated as the number of years infected during follow-up. Multivariable logistic regression models were used to estimate the risk of progression to a more advanced diagnosis (versus no change/regression) as well as gastric cancer risk by intestinal metaplasia (IM) subtype. For a more detailed analysis of progression, we also used a histopathology score assessing both severity and extension of the gastric lesions (range 1-6). The score difference between baseline and 16 years was modelled by generalised linear models. RESULTS: Individuals who were continuously infected with H. pylori for 16 years had a higher probability of progression to a more advanced diagnosis than those who cleared the infection and remained negative after baseline (p=0.001). Incomplete-type IM was associated with higher risk of progression to cancer than complete-type (OR, 11.3; 95% CI 1.4 to 91.4). The average histopathology score increased by 0.20 units/year (95% CI 0.12 to 0.28) among individuals continuously infected with H. pylori. The effect of cumulative time of infection on progression in the histopathology score was significantly higher for individuals with atrophy (without IM) than for individuals with IM (p<0.001). CONCLUSIONS: Long-term exposure to H. pylori infection was associated with progression of precancerous lesions. Individuals infected with H. pylori with these lesions may benefit from eradication, particularly those with atrophic gastritis without IM. Incomplete-type IM may be a useful marker for the identification of individuals at higher risk for cancer.

The L-Arginine Transporter Solute Carrier Family 7 Member 2 Mediates the Immunopathogenesis of Attaching and Effacing Bacteria.

Solute carrier family 7 member 2 (SLC7A2) is an inducible transporter of the semi-essential amino acid L-arginine (L-Arg), which has been implicated in immune responses to pathogens. We assessed the role of SLC7A2 in murine infection with Citrobacter rodentium, an attaching and effacing enteric pathogen that causes colitis. Induction of SLC7A2 was upregulated in colitis tissues, and localized predominantly to colonic epithelial cells. Compared to wild-type mice, Slc7a2-/-mice infected with C. rodentium had improved survival and decreased weight loss, colon weight, and histologic injury; this was associated with decreased colonic macrophages, dendritic cells, granulocytes, and Th1 and Th17 cells. In infected Slc7a2-/-mice, there were decreased levels of the proinflammatory cytokines G-CSF, TNF-α, IL-1α, IL-1ß, and the chemokines CXCL1, CCL2, CCL3, CCL4, CXCL2, and CCL5. In bone marrow chimeras, the recipient genotype drove the colitis phenotype, indicative of the importance of epithelial, rather than myeloid SLC7A2. Mice lacking Slc7a2 exhibited reduced adherence of C. rodentium to the colonic epithelium and decreased expression of Talin-1, a focal adhesion protein involved in the attachment of the bacterium. The importance of SLC7A2 and Talin-1 in the intimate attachment of C. rodentium and induction of inflammatory response was confirmed in vitro, using conditionally-immortalized young adult mouse colon (YAMC) cells with shRNA knockdown of Slc7a2 or Tln1. Inhibition of L-Arg uptake with the competitive inhibitor, L-lysine (L-Lys), also prevented attachment of C. rodentium and chemokine expression. L-Lys and siRNA knockdown confirmed the role of L-Arg and SLC7A2 in human Caco-2 cells co-cultured with enteropathogenic Escherichia coli. Overexpression of SLC7A2 in human embryonic kidney cells increased bacterial adherence and chemokine expression. Taken together, our data indicate that C. rodentium enhances its own pathogenicity by inducing the expression of SLC7A2 to favor its attachment to the epithelium and thus create its ecological niche.

TLR9 activation suppresses inflammation in response to Helicobacter pylori infection.

Helicobacter pylori (H. pylori) induces chronic gastritis in humans, and infection can persist for decades. One H. pylori strain-specific constituent that augments disease risk is the cag pathogenicity island. The cag island encodes a type IV secretion system (T4SS) that translocates DNA into host cells. Toll-like receptor 9 (TLR9) is an innate immune receptor that detects hypo-methylated CpG DNA motifs. In this study, we sought to define the role of the H. pylori cag T4SS on TLR9-mediated responses in vivo. H. pylori strain PMSS1 or its cagE- mutant, which fails to assemble a T4SS, were used to infect wild-type or Tlr9-/- C57BL/6 mice. PMSS1-infected Tlr9-/- mice developed significantly higher levels of inflammation, despite similar levels of colonization density, compared with PMSS1-infected wild-type mice. These changes were cag dependent, as both mouse genotypes infected with the cagE- mutant only developed minimal inflammation. Tlr9-/- genotypes did not alter the microbial phenotypes of in vivo-adapted H. pylori strains; therefore, we examined host immunological responses. There were no differences in levels of TH1 or TH2 cytokines in infected mice when stratified by host genotype. However, gastric mucosal levels of IL-17 were significantly increased in infected Tlr9-/- mice compared with infected wild-type mice, and H. pylori infection of IL-17A-/- mice concordantly led to significantly decreased levels of gastritis. Thus loss of Tlr9 selectively augments the intensity of IL-17-driven immune responses to H. pylori in a cag T4SS-dependent manner. These results suggest that H. pylori utilizes the cag T4SS to manipulate the intensity of the host immune response.

Epigenetic and genetic variation in GATA5 is associated with gastric disease risk.

Gastric cancer incidence varies considerably among populations, even those with comparable rates of Helicobacter pylori infection. To test the hypothesis that genetic variation plays a role in gastric disease, we assessed the relationship between genotypes and gastric histopathology in a Colombian study population, using a genotyping array of immune-related single nucleotide polymorphisms (SNPs). Two synonymous SNPs (rs6061243 and rs6587239) were associated with progression of premalignant gastric lesions in a dominant-effects model after correction for multiple comparisons (p = 2.63E-07 and p = 7.97E-07, respectively); effect sizes were ß = -0.863 and ß = -0.815, respectively, where ß is an estimate of effect on histopathology scores, which ranged from 1 (normal) to 5 (dysplasia). In our replication cohort, a second Colombian population, both SNPs were associated with histopathology when additively modeled (ß = -0.256, 95 % CI = -0.47, -0.039; and ß = -0.239, 95 % CI = -0.45, -0.024), and rs6587239 was significantly associated in a dominant-effects model (ß = -0.330, 95 % CI = -0.66, 0.00). Because promoter methylation of GATA5 has previously been associated with gastric cancer, we also tested for the association of methylation status with more advanced histopathology scores in our samples and found a significant relationship (p = 0.001). A multivariate regression model revealed that the effects of both the promoter methylation and the exonic SNPs in GATA5 were independent. A SNP-by-methylation interaction term was also significant. This interaction between GATA5 variants and GATA5 promoter methylation indicates that the association of either factor with gastric disease progression is modified by the other.

The host protein calprotectin modulates the Helicobacter pylori cag type IV secretion system via zinc sequestration.

Transition metals are necessary for all forms of life including microorganisms, evidenced by the fact that 30% of all proteins are predicted to interact with a metal cofactor. Through a process termed nutritional immunity, the host actively sequesters essential nutrient metals away from invading pathogenic bacteria. Neutrophils participate in this process by producing several metal chelating proteins, including lactoferrin and calprotectin (CP). As neutrophils are an important component of the inflammatory response directed against the bacterium Helicobacter pylori, a major risk factor for gastric cancer, it was hypothesized that CP plays a role in the host response to H. pylori. Utilizing a murine model of H. pylori infection and gastric epithelial cell co-cultures, the role CP plays in modifying H. pylori -host interactions and the function of the cag Type IV Secretion System (cag T4SS) was investigated. This study indicates elevated gastric levels of CP are associated with the infiltration of neutrophils to the H. pylori-infected tissue. When infected with an H. pylori strain harboring a functional cag T4SS, calprotectin-deficient mice exhibited decreased bacterial burdens and a trend toward increased cag T4SS -dependent inflammation compared to wild-type mice. In vitro data demonstrate that culturing H. pylori with sub-inhibitory doses of CP reduces the activity of the cag T4SS and the biogenesis of cag T4SS-associated pili in a zinc-dependent fashion. Taken together, these data indicate that zinc homeostasis plays a role in regulating the proinflammatory activity of the cag T4SS.

Heme oxygenase-1 dysregulates macrophage polarization and the immune response to Helicobacter pylori.

Helicobacter pylori incites a futile inflammatory response, which is the key feature of its immunopathogenesis. This leads to the ability of this bacterial pathogen to survive in the stomach and cause peptic ulcers and gastric cancer. Myeloid cells recruited to the gastric mucosa during H. pylori infection have been directly implicated in the modulation of host defense against the bacterium and gastric inflammation. Heme oxygenase-1 (HO-1) is an inducible enzyme that exhibits anti-inflammatory functions. Our aim was to analyze the induction and role of HO-1 in macrophages during H. pylori infection. We now show that phosphorylation of the H. pylori virulence factor cytotoxin-associated gene A (CagA) in macrophages results in expression of hmox-1, the gene encoding HO-1, through p38/NF (erythroid-derived 2)-like 2 signaling. Blocking phagocytosis prevented CagA phosphorylation and HO-1 induction. The expression of HO-1 was also increased in gastric mononuclear cells of human patients and macrophages of mice infected with cagA(+) H. pylori strains. Genetic ablation of hmox-1 in H. pylori-infected mice increased histologic gastritis, which was associated with enhanced M1/Th1/Th17 responses, decreased regulatory macrophage (Mreg) response, and reduced H. pylori colonization. Gastric macrophages of H. pylori-infected mice and macrophages infected in vitro with this bacterium showed an M1/Mreg mixed polarization type; deletion of hmox-1 or inhibition of HO-1 in macrophages caused an increased M1 and a decrease of Mreg phenotype. These data highlight a mechanism by which H. pylori impairs the immune response and favors its own survival via activation of macrophage HO-1.

The Human Antimicrobial Protein Calgranulin C Participates in Control of Helicobacter pylori Growth and Regulation of Virulence.

During infectious processes, antimicrobial proteins are produced by both epithelial cells and innate immune cells. Some of these antimicrobial molecules function by targeting transition metals and sequestering these metals in a process referred to as "nutritional immunity." This chelation strategy ultimately starves invading pathogens, limiting their growth within the vertebrate host. Recent evidence suggests that these metal-binding antimicrobial molecules have the capacity to affect bacterial virulence, including toxin secretion systems. Our previous work showed that the S100A8/S100A9 heterodimer (calprotectin, or calgranulin A/B) binds zinc and represses the elaboration of the H. pylori cag type IV secretion system (T4SS). However, there are several other S100 proteins that are produced in response to infection. We hypothesized that the zinc-binding protein S100A12 (calgranulin C) is induced in response to H. pylori infection and also plays a role in controlling H. pylori growth and virulence. To test this, we analyzed gastric biopsy specimens from H. pylori-positive and -negative patients for S100A12 expression. These assays showed that S100A12 is induced in response to H. pylori infection and inhibits bacterial growth and viability in vitro by binding nutrient zinc. Furthermore, the data establish that the zinc-binding activity of the S100A12 protein represses the activity of the cag T4SS, as evidenced by the gastric cell "hummingbird" phenotype, interleukin 8 (IL-8) secretion, and CagA translocation assays. In addition, high-resolution field emission gun scanning electron microscopy (FEG-SEM) was used to demonstrate that S100A12 represses biogenesis of the cag T4SS. Together with our previous work, these data reveal that multiple S100 proteins can repress the elaboration of an oncogenic bacterial surface organelle.

Activation of EGFR and ERBB2 by Helicobacter pylori results in survival of gastric epithelial cells with DNA damage.

BACKGROUND & AIMS: The gastric cancer-causing pathogen Helicobacter pylori up-regulates spermine oxidase (SMOX) in gastric epithelial cells, causing oxidative stress-induced apoptosis and DNA damage. A subpopulation of SMOX(high) cells are resistant to apoptosis, despite their high levels of DNA damage. Because epidermal growth factor receptor (EGFR) activation can regulate apoptosis, we determined its role in SMOX-mediated effects. METHODS: SMOX, apoptosis, and DNA damage were measured in gastric epithelial cells from H. pylori-infected Egfr(wa5) mice (which have attenuated EGFR activity), Egfr wild-type mice, or in infected cells incubated with EGFR inhibitors or deficient in EGFR. A phosphoproteomic analysis was performed. Two independent tissue microarrays containing each stage of disease, from gastritis to carcinoma, and gastric biopsy specimens from Colombian and Honduran cohorts were analyzed by immunohistochemistry. RESULTS: SMOX expression and DNA damage were decreased, and apoptosis increased in H. pylori-infected Egfr(wa5) mice. H. pylori-infected cells with deletion or inhibition of EGFR had reduced levels of SMOX, DNA damage, and DNA damage(high) apoptosis(low) cells. Phosphoproteomic analysis showed increased EGFR and erythroblastic leukemia-associated viral oncogene B (ERBB)2 signaling. Immunoblot analysis showed the presence of a phosphorylated (p)EGFR-ERBB2 heterodimer and pERBB2; knockdown of ErbB2 facilitated apoptosis of DNA damage(high) apoptosis(low) cells. SMOX was increased in all stages of gastric disease, peaking in tissues with intestinal metaplasia, whereas pEGFR, pEGFR-ERBB2, and pERBB2 were increased predominantly in tissues showing gastritis or atrophic gastritis. Principal component analysis separated gastritis tissues from patients with cancer vs those without cancer. pEGFR, pEGFR-ERBB2, pERBB2, and SMOX were increased in gastric samples from patients whose disease progressed to intestinal metaplasia or dysplasia, compared with patients whose disease did not progress. CONCLUSIONS: In an analysis of gastric tissues from mice and patients, we identified a molecular signature (based on levels of pEGFR, pERBB2, and SMOX) for the initiation of gastric carcinogenesis.

The nutraceutical electrophile scavenger 2-hydroxybenzylamine (2-HOBA) attenuates gastric cancer development caused by Helicobacter pylori.

Stomach cancer is a leading cause of cancer death. Helicobacter pylori is a bacterial gastric pathogen that is the primary risk factor for carcinogenesis, associated with its induction of inflammation and DNA damage. Dicarbonyl electrophiles are generated from lipid peroxidation during the inflammatory response and form covalent adducts with amine-containing macromolecules. 2-hydroxybenzylamine (2-HOBA) is a natural compound derived from buckwheat seeds and acts as a potent scavenger of reactive aldehydes. Our goal was to investigate the effect of 2-HOBA on the pathogenesis of H. pylori infection. We used transgenic FVB/N insulin-gastrin (INS-GAS) mice as a model of gastric cancer. First, we found that 2-HOBA is bioavailable in the gastric tissues of these mice after supplementation in the drinking water. Moreover, 2-HOBA reduced the development of gastritis in H. pylori-infected INS-GAS mice without affecting the bacterial colonization level in the stomach. Further, we show that the development of gastric dysplasia and carcinoma was significantly reduced by 2-HOBA. Concomitantly, DNA damage were also inhibited by 2-HOBA treatment in H. pylori-infected mice. In parallel, DNA damage was inhibited by 2-HOBA in H. pylori-infected gastric epithelial cells in vitro. In conclusion, 2-HOBA, which has been shown to be safe in human clinical trials, represents a promising nutritional compound for the chemoprevention of the more severe effects of H. pylori infection.

Epithelial talin-1 protects mice from citrobacter rodentium-induced colitis by restricting bacterial crypt intrusion and enhancing t cell immunity.

Pathogenic enteric Escherichia coli present a significant burden to global health. Food-borne enteropathogenic E. coli (EPEC) and Shiga toxin-producing E. coli (STEC) utilize attaching and effacing (A/E) lesions and actin-dense pedestal formation to colonize the gastrointestinal tract. Talin-1 is a large structural protein that links the actin cytoskeleton to the extracellular matrix though direct influence on integrins. Here we show that mice lacking talin-1 in intestinal epithelial cells (Tln1Δepi) have heightened susceptibility to colonic disease caused by the A/E murine pathogen Citrobacter rodentium. Tln1Δepi mice exhibit decreased survival, and increased colonization, colon weight, and histologic colitis compared to littermate Tln1fl/fl controls. These findings were associated with decreased actin polymerization and increased infiltration of innate myeloperoxidase-expressing immune cells, confirmed as neutrophils by flow cytometry, but more bacterial dissemination deep into colonic crypts. Further evaluation of the immune population recruited to the mucosa in response to C. rodentium revealed that loss of Tln1 in colonic epithelial cells (CECs) results in impaired recruitment and activation of T cells. C. rodentium infection-induced colonic mucosal hyperplasia was exacerbated in Tln1Δepi mice compared to littermate controls. We demonstrate that this is associated with decreased CEC apoptosis and crowding of proliferating cells in the base of the glands. Taken together, talin-1 expression by CECs is important in the regulation of both epithelial renewal and the inflammatory T cell response in the setting of colitis caused by C. rodentium, suggesting that this protein functions in CECs to limit, rather than contribute to the pathogenesis of this enteric infection.

Targeting hypoxia-inducible factor-1 alpha suppresses Helicobacter pylori-induced gastric injury via attenuation of both cag-mediated microbial virulence and proinflammatory host responses.

Helicobacter pylori-induced inflammation is the strongest known risk factor for gastric adenocarcinoma. Hypoxia-inducible factor-1 (HIF-1α) is a key transcriptional regulator of immunity and carcinogenesis. To examine the role of this mediator within the context of H. pylori-induced injury, we first demonstrated that HIF-1α levels were significantly increased in parallel with the severity of gastric lesions in humans. In interventional studies targeting HIF-1α, H. pylori-infected mice were treated ± dimethyloxalylglycine (DMOG), a prolyl hydroxylase inhibitor that stabilizes HIF-1α. H. pylori significantly increased proinflammatory chemokines/cytokines and inflammation in vehicle-treated mice; however, this was significantly attenuated in DMOG-treated mice. DMOG treatment also significantly decreased function of the H. pylori type IV secretion system (T4SS) in vivo and significantly reduced T4SS-mediated NF-κB activation and IL-8 induction in vitro. These results suggest that prolyl hydroxylase inhibition protects against H. pylori-mediated pathologic responses, and is mediated, in part, via attenuation of H. pylori cag-mediated virulence and suppression of host proinflammatory responses.