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Helicobacter pylori is the primary cause of gastric cancer, and there is a need to discover new molecular targets for therapeutic intervention in H. pylori disease progression. We have previously shown that spermine oxidase (SMOX), the enzyme that catabolizes the back-conversion of the polyamine spermine to spermidine, is upregulated during infection and is associated with increased cancer risk in humans. We sought to determine the direct role of SMOX in gastric carcinogenesis during H. pylori infection. In this study, we demonstrate that transgenic FVB/N insulin-gastrin (INS-GAS) mice that develop gastric carcinoma with H. pylori infection were protected from cancer development with Smox deletion. RNA sequencing revealed that genes associated with the immune system and cancer were downregulated in the infected Smox -/- mice. Furthermore, there was a decrease in cell proliferation and DNA damage in infected Smox -/- animals. There was significant generation of adducts of the highly reactive electrophile acrolein, a byproduct of SMOX activity, in gastric tissues from H. pylori-infected humans and wild-type, but not Smox -/- mice. Genetic deletion of Smox in murine organoids or chemical inhibition of SMOX in human gastric epithelial cells significantly reduced generation of acrolein induced by H. pylori. Additionally, acrolein-induced DNA damage in gastric epithelial cells was ablated with the electrophile scavenger 2-hydroxybenzylamine (2-HOBA). Gastric acrolein adduct levels were attenuated in infected INS-GAS mice treated with 2-HOBA, which exhibit reduced gastric carcinoma. These findings implicate SMOX and acrolein in H. pylori-induced carcinogenesis, thus indicating their potential as therapeutic targets., Competing Interests: Competing interests APG and KTW are named inventors on a US patent, Compounds and Methods for Scavenging Dicarbonyl Electrophiles (Patent # 11696903), for use of electrophile scavengers, which is assigned to Vanderbilt University and The United States Government as represented by the Department of Veterans Affairs. In addition, APG and KTW receive royalty payments from a licensing agreement between Vanderbilt University and MTI Biotech for the future use of electrophile scavengers. All other authors have declared that no conflict of interest exists., (© 2024. This is a U.S. Government work and not under copyright protection in the US; foreign copyright protection may apply.)

Cystathionine γ-lyase (CTH) is a critical enzyme in the reverse transsulfuration pathway, the major route for the metabolism of sulfur-containing amino acids, notably converting cystathionine to cysteine. We reported that CTH supports gastritis induced by the pathogen Helicobacter pylori. Herein our aim was to investigate the role of CTH in colonic inflammation. First, we found that CTH is induced in the colon mucosa in mice with dextran sulfate sodium-induced colitis. Expression of CTH was completely absent in the colon of Cth -/- mice. We observed that clinical and histological parameters are ameliorated in Cth-deficient mice compared to wild-type animals. However, Cth deletion had no effect on tumorigenesis and the level of dysplasia in mice treated with azoxymethane-DSS, as a reliable model of colitis-associated carcinogenesis. Mechanistically, we determined that the deletion of the gene Slc7a11 encoding for solute carrier family 7 member 11, the transporter of the anionic form of cysteine, does not affect DSS colitis. Lastly, we found that the richness and diversity of the fecal microbiota were significantly increased in Cth -/- mice compared to both WT and Slc7a11 -/- mice. In conclusion, our data suggest that the enzyme CTH represents a target for clinical intervention in patients with inflammatory bowel disease, potentially by beneficially reshaping the composition of the gut microbiota., (© 2024. This is a U.S. Government work and not under copyright protection in the US; foreign copyright protection may apply.)

Crohn's disease (CD) is a complex chronic inflammatory disorder with both gastrointestinal and extra-intestinal manifestations associated immune dysregulation. Analyzing 202,359 cells from 170 specimens across 83 patients, we identify a distinct epithelial cell type in both terminal ileum and ascending colon (hereon as 'LND') with high expression of LCN2, NOS2, and DUOX2 and genes related to antimicrobial response and immunoregulation. LND cells, confirmed by in-situ RNA and protein imaging, are rare in non-IBD controls but expand in active CD, and actively interact with immune cells and specifically express IBD/CD susceptibility genes, suggesting a possible function in CD immunopathogenesis. Furthermore, we discover early and late LND subpopulations with different origins and developmental potential. A higher ratio of late-to-early LND cells correlates with better response to anti-TNF treatment. Our findings thus suggest a potential pathogenic role for LND cells in both Crohn's ileitis and colitis., (© 2024. The Author(s).)

Hypusine is an amino acid synthesized by the enzyme deoxyhypusine synthase (DHPS). It is critical for the activity of eukaryotic translation initiation factor 5A (EIF5A). We reported that hypusination i ) in macrophages supports the innate response towards pathogenic bacteria and ii ) in epithelial cells maintains intestinal homeostasis. Herein, we investigated the effect of myeloid hypusination on the outcome of colitis and colitis-associated cancer. We found that patients with Crohn's disease exhibit increased levels of DHPS and EIF5A Hyp in cells infiltrating the colon lamina propria. However, the specific deletion of Dhps in myeloid cells had no impact on clinical, histological, or inflammatory parameters in mice treated with dextran sulfate sodium (DSS). Further, tumorigenesis and level of dysplasia were not affected by myeloid deletion of Dhps in the azoxymethane-DSS model. The composition of the fecal and the mucosa-associated microbiome was similar in animals lacking or not DHPS in myeloid cells. Thus, hypusination in myeloid cells does not regulate colitis associated with epithelial injury and colitis-associated cancer. Enhancement of the DHPS/hypusine pathway in patients with inflammatory bowel disease could have therapeutic impact through epithelial effects, but modulation of hypusination in myeloid cells will be unlikely to affect the disease., Competing Interests: 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.

The intestinal immune response is crucial in maintaining a healthy gut, but the enhanced migration of macrophages in response to pathogens is a major contributor to disease pathogenesis. Integrins are ubiquitously expressed cellular receptors that are highly involved in immune cell adhesion to endothelial cells while in the circulation and help facilitate extravasation into tissues. Here we show that specific deletion of the Tln1 gene encoding the protein talin-1, an integrin-activating scaffold protein, from cells of the myeloid lineage using the Lyz2-cre driver mouse reduces epithelial damage, attenuates colitis, downregulates the expression of macrophage markers, decreases the number of differentiated colonic mucosal macrophages, and diminishes the presence of CD68-positive cells in the colonic mucosa of mice infected with the enteric pathogen Citrobacter rodentium. Bone marrow-derived macrophages lacking expression of Tln1 did not exhibit a cell-autonomous phenotype; there was no impaired proinflammatory gene expression, nitric oxide production, phagocytic ability, or surface expression of CD11b, CD86, or major histocompatibility complex II in response to C. rodentium. Thus, we demonstrate that talin-1 plays a role in the manifestation of infectious colitis by increasing mucosal macrophages, with an effect that is independent of macrophage activation., (© 2023. This is a U.S. Government work and not under copyright protection in the US; foreign copyright protection may apply.)

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 (EIF5A Hyp ) remains unknown in intestinal homeostasis. Our aim was to investigate EIF5A Hyp 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 EIF5A Hyp . 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., (Published by Elsevier Inc.)

Colorectal cancer (CRC) is a major health problem worldwide. Dicarbonyl electrophiles, such as isolevuglandins (isoLGs), are generated from lipid peroxidation and form covalent adducts with amine-containing macromolecules. We have shown high levels of adducts of isoLGs in colonic epithelial cells of patients with CRC. We thus investigated the role of these reactive aldehydes in colorectal cancer development. We found that 2-hydroxybenzylamine (2-HOBA), a natural compound derived from buckwheat seeds that acts as a potent scavenger of electrophiles, is bioavailable in the colon of mice after supplementation in the drinking water and does not affect the colonic microbiome. 2-HOBA reduced the level of isoLG adducts to lysine as well as tumorigenesis in models of colitis-associated carcinogenesis and of sporadic CRC driven by specific deletion of the adenomatous polyposis coli gene in colonic epithelial cells. In parallel, we found that oncogenic NRF2 activation and signaling were decreased in the colon of 2-HOBA-treated mice. Additionally, the growth of xenografted human HCT116 CRC cells in nude mice was significantly attenuated by 2-HOBA supplementation. In conclusion, 2-HOBA represents a promising natural compound for the prevention and treatment of CRC., (© 2023. This is a U.S. Government work and not under copyright protection in the US; foreign copyright protection may apply.)

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., Competing Interests: Declaration of Competing Interest The authors declare the following financial interests/personal relationships which may be considered as potential competing interests: Keith T. Wilson reports financial support was provided by National Cancer Institute. John A. Rathmacher reports financial support was provided by National Cancer Institute. Keith T. Wilson reports financial support was provided by US Department of Defense. Keith T. Wilson reports financial support was provided by US Department of Veterans Affairs. Keith T. Wilson reports financial support was provided by National Institute of Diabetes and Digestive and Kidney Diseases. Keith T. Wilson reports a relationship with MTI Biotech that includes: non-financial support. Keith T. Wilson has patent #16/893,425 pending to Assignee. Keith T. Wilson and John A. Rathmacher are co-principal investigators on NIH grant R41CA257262. John Rathmacher is employed by MTI Biotech. Keith T. Wilson and Alain P. Gobert are names as inventors on US patent application 16/893.425. This patent application is still in the review process. MTI Biotech has a licensing agreement with Vanderbilt University for future use of electrophile scavenger drugs, include the 2-Hydroxybenzylamine used in the current manuscript. Dr. Wilson and Dr. Gobert are named as potential recipients of royalty payments, although none have been received to date., (Published by Elsevier Masson SAS.)

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 ( Tln1 Δepi ) have heightened susceptibility to colonic disease caused by the A/E murine pathogen Citrobacter rodentium . Tln1 Δepi mice exhibit decreased survival, and increased colonization, colon weight, and histologic colitis compared to littermate Tln1 fl/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 Tln1 Δepi 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.

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., (Copyright © 2022 by The American Association of Immunologists, Inc.)

Macrophages play a crucial role in the inflammatory response to the human stomach pathogen Helicobacter pylori, which infects half of the world's population and causes gastric cancer. Recent studies have highlighted the importance of macrophage immunometabolism in their activation state and function. We have demonstrated that the cysteine-producing enzyme cystathionine γ-lyase (CTH) is upregulated in humans and mice with H. pylori infection. Here, we show that induction of CTH in macrophages by H. pylori promoted persistent inflammation. Cth-/- mice had reduced macrophage and T cell activation in H. pylori-infected tissues, an altered metabolome, and decreased enrichment of immune-associated gene networks, culminating in decreased H. pylori-induced gastritis. CTH is downstream of the proposed antiinflammatory molecule, S-adenosylmethionine (SAM). Whereas Cth-/- mice exhibited gastric SAM accumulation, WT mice treated with SAM did not display protection against H. pylori-induced inflammation. Instead, we demonstrated that Cth-deficient macrophages exhibited alterations in the proteome, decreased NF-κB activation, diminished expression of macrophage activation markers, and impaired oxidative phosphorylation and glycolysis. Thus, through altering cellular respiration, CTH is a key enhancer of macrophage activation, contributing to a pathogenic inflammatory response that is the universal precursor for the development of H. pylori-induced gastric disease.

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., (Published by Elsevier Inc.)

Gastric cancer (GC) is the fifth most common cancer and the fourth most common cause of cancer-related death worldwide. The intestinal type of GC progresses from acute to chronic gastritis, multifocal atrophic gastritis, intestinal metaplasia, dysplasia, and carcinoma. Infection of the stomach by Helicobacter pylori, a Gram-negative bacterium that infects approximately 50% of the world's population, is the causal determinant that initiates the gastric inflammation and then disease progression. In this context, the induction of the innate immune response of gastric epithelial cells and myeloid cells by H. pylori effectors plays a critical role in the outcome of the infection. However, only 1% to 3% of infected patients develop gastric adenocarcinoma, emphasizing that other mechanisms regulate the localized non-specific response, including the gastric microbiota and genetic factors. This review summarizes studies describing the factors that induce and regulate the mucosal innate immune response during H. pylori infection., (Copyright © 2022 The Authors. Published by Elsevier Inc. All rights reserved.)

CCL11, also known as eotaxin-1, is described as an eosinophil chemoattractant, which has been implicated in allergic and Th2 inflammatory diseases. We have reported that CCL11 is significantly increased in the serum of inflammatory bowel disease (IBD) patients, colonic eosinophils are increased and correlate with tissue CCL11 levels in ulcerative colitis patients, and CCL11 is increased in dextran sulfate sodium (DSS)-induced murine colitis. Here, we show that CCL11 is involved in the pathogenesis of DSS-induced colitis and in colon tumorigenesis in the azoxymethane (AOM)-DSS model of colitis-associated carcinogenesis (CAC). Ccl11 -/- mice exposed to DSS then allowed to recover had significantly less body weight loss and a decrease in histologic injury versus wild-type (WT) mice. In the AOM-DSS model, Ccl11 -/- mice exhibited decreased colonic tumor number and burden, histologic injury, and colonic eosinophil infiltration versus WT mice. Ccl11 is expressed by both colonic epithelial and lamina propria immune cells. Studies in bone marrow chimera mice revealed that hematopoietic- and epithelial-cell-derived CCL11 were both important for tumorigenesis in the AOM-DSS model. These findings indicate that CCL11 is important in the regulation of colitis and associated carcinogenesis and thus anti-CCL11 antibodies may be useful for treatment and cancer chemoprevention in IBD., (© 2021. This is a U.S. government work and not under copyright protection in the U.S.; foreign copyright protection may apply.)

Advancements in our understanding of polyamine molecular and cellular functions have led to increased interest in targeting polyamine metabolism for anticancer therapeutic benefits. The polyamines putrescine, spermidine, and spermine are polycationic alkylamines commonly found in all living cells and are essential for cellular growth and survival. This review summarizes the existing research on polyamine metabolism and function, specifically the role of polyamines in gastric immune cell and epithelial cell function. Polyamines have been implicated in a multitude of cancers, but in this review, we focus on the role of polyamine dysregulation in the context of Helicobacter pylori-induced gastritis and subsequent progression to gastric cancer. Due to the emerging implication of polyamines in cancer development, there is an increasing number of promising clinical trials using agents to target the polyamine metabolic pathway for potential chemoprevention and anticancer therapy.

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., (Published by Elsevier Inc.)

Innate responses of myeloid cells defend against pathogenic bacteria via inducible effectors. Deoxyhypusine synthase (DHPS) catalyzes the transfer of the N-moiety of spermidine to the lysine-50 residue of eukaryotic translation initiation factor 5A (EIF5A) to form the amino acid hypusine. Hypusinated EIF5A (EIF5A Hyp ) transports specific mRNAs to ribosomes for translation. We show that DHPS is induced in macrophages by two gastrointestinal pathogens, Helicobacter pylori and Citrobacter rodentium, resulting in enhanced hypusination of EIF5A. EIF5A Hyp was also increased in gastric macrophages from patients with H. pylori gastritis. Furthermore, we identify the bacteria-induced immune effectors regulated by hypusination. This set of proteins includes essential constituents of antimicrobial response and autophagy. Mice with myeloid cell-specific deletion of Dhps exhibit reduced EIF5A Hyp in macrophages and increased bacterial burden and inflammation. Thus, regulation of translation through hypusination is a critical hallmark of the defense of eukaryotic hosts against pathogenic bacteria., Competing Interests: Declaration of Interests K.T.W. serves as an associate editor for the journal Gastroenterology. He receives compensation for this work from the American Gastroenterological Association. This position is not related to this study., (Published by Elsevier Inc.)

Helicobacter pylori infection is the main risk factor for the development of gastric cancer, the third leading cause of cancer death worldwide. H. pylori colonizes the human gastric mucosa and persists for decades. The inflammatory response is ineffective in clearing the infection, leading to disease progression that may result in gastric adenocarcinoma. We have shown that polyamines are regulators of the host response to H. pylori, and that spermine oxidase (SMOX), which metabolizes the polyamine spermine into spermidine plus H 2 O 2 , is associated with increased human gastric cancer risk. We now used a molecular approach to directly address the role of SMOX, and demonstrate that Smox-deficient mice exhibit significant reductions of gastric spermidine levels and H. pylori-induced inflammation. Proteomic analysis revealed that cancer was the most significantly altered functional pathway in Smox -/- gastric organoids. Moreover, there was also less DNA damage and β-catenin activation in H. pylori-infected Smox -/- mice or gastric organoids, compared to infected wild-type animals or gastroids. The link between SMOX and β-catenin activation was confirmed in human gastric organoids that were treated with a novel SMOX inhibitor. These findings indicate that SMOX promotes H. pylori-induced carcinogenesis by causing inflammation, DNA damage, and activation of β-catenin signaling.

Naturally occurring polyamines are ubiquitously distributed and play important roles in cell development, amino acid and protein synthesis, oxidative DNA damage, proliferation, and cellular differentiation. Macrophages are essential in the innate immune response, and contribute to tissue remodeling. Naïve macrophages have two major potential fates: polarization to (1) the classical pro-inflammatory M1 defense response to bacterial pathogens and tumor cells, and (2) the alternatively activated M2 response, induced in the presence of parasites and wounding, and also implicated in the development of tumor-associated macrophages. ODC, the rate-limiting enzyme in polyamine synthesis, leads to an increase in putrescine levels, which impairs M1 gene transcription. Additionally, spermidine and spermine can regulate translation of pro-inflammatory mediators in activated macrophages. In this review, we focus on polyamines in macrophage activation patterns in the context of gastrointestinal inflammation and carcinogenesis. We seek to clarify mechanisms of innate immune regulation by polyamine metabolism and potential novel therapeutic targets.

The reverse transsulfuration pathway is the major route for the metabolism of sulfur-containing amino acids. The role of this metabolic pathway in macrophage response and function is unknown. We show that the enzyme cystathionine γ-lyase (CTH) is induced in macrophages infected with pathogenic bacteria through signaling involving phosphatidylinositol 3-kinase (PI3K)/MTOR and the transcription factor SP1. This results in the synthesis of cystathionine, which facilitates the survival of pathogens within myeloid cells. Our data demonstrate that the expression of CTH leads to defective macrophage activation by (i) dysregulation of polyamine metabolism by depletion of S -adenosylmethionine, resulting in immunosuppressive putrescine accumulation and inhibition of spermidine and spermine synthesis, and (ii) increased histone H3K9, H3K27, and H3K36 di/trimethylation, which is associated with gene expression silencing. Thus, CTH is a pivotal enzyme of the innate immune response that disrupts host defense. The induction of the reverse transsulfuration pathway by bacterial pathogens can be considered an unrecognized mechanism for immune escape. IMPORTANCE Macrophages are professional immune cells that ingest and kill microbes. In this study, we show that different pathogenic bacteria induce the expression of cystathionine γ-lyase (CTH) in macrophages. This enzyme is involved in a metabolic pathway called the reverse transsulfuration pathway, which leads to the production of numerous metabolites, including cystathionine. Phagocytized bacteria use cystathionine to better survive in macrophages. In addition, the induction of CTH results in dysregulation of the metabolism of polyamines, which in turn dampens the proinflammatory response of macrophages. In conclusion, pathogenic bacteria can evade the host immune response by inducing CTH in macrophages.

There is great interest in safe and effective alternative therapies that could benefit patients with inflammatory bowel diseases (IBD). L-arginine (Arg) is a semi-essential amino acid with a variety of physiological effects. In this context, our aim was to investigate the role of dietary Arg in experimental colitis. We used two models of colitis in C57BL/6 mice, the dextran sulfate sodium (DSS) model of injury and repair, and Citrobacter rodentium infection. Animals were given diets containing (1) no Arg (Arg 0 ), 6.4 g/kg (Arg NL ), or 24.6 g/kg Arg (Arg HIGH ); or (2) the amino acids downstream of Arg: 28 g/kg L-ornithine (Orn HIGH ) or 72 g/kg L-proline (Pro HIGH ). Mice with DSS colitis receiving the Arg HIGH diet had increased levels of Arg, Orn, and Pro in the colon and improved body weight loss, colon length shortening, and histological injury compared to Arg NL and Arg 0 diets. Histology was improved in the Arg NL vs. Arg 0 group. Orn HIGH or Pro HIGH diets did not provide protection. Reduction in colitis with Arg HIGH diet also occurred in C. rodentium -infected mice. Diversity of the intestinal microbiota was significantly enhanced in mice on the Arg HIGH diet compared to the Arg NL or Arg 0 diets, with increased abundance of Bacteroidetes and decreased Verrucomicrobia. In conclusion, dietary supplementation of Arg is protective in colitis models. This may occur by restoring overall microbial diversity and Bacteroidetes prevalence. Our data provide a rationale for Arg as an adjunctive therapy in IBD.

Infection by Helicobacter pylori is the primary cause of gastric adenocarcinoma. The most potent H. pylori virulence factor is cytotoxin-associated gene A (CagA), which is translocated by a type 4 secretion system (T4SS) into gastric epithelial cells and activates oncogenic signaling pathways. The gene cagY encodes for a key component of the T4SS and can undergo gene rearrangements. We have shown that the cancer chemopreventive agent α-difluoromethylornithine (DFMO), known to inhibit the enzyme ornithine decarboxylase, reduces H. pylori -mediated gastric cancer incidence in Mongolian gerbils. In the present study, we questioned whether DFMO might directly affect H. pylori pathogenicity. We show that H. pylori output strains isolated from gerbils treated with DFMO exhibit reduced ability to translocate CagA in gastric epithelial cells. Further, we frequently detected genomic modifications in the middle repeat region of the cagY gene of output strains from DFMO-treated animals, which were associated with alterations in the CagY protein. Gerbils did not develop carcinoma when infected with a DFMO output strain containing rearranged cagY or the parental strain in which the wild-type cagY was replaced by cagY with DFMO-induced rearrangements. Lastly, we demonstrate that in vitro treatment of H. pylori by DFMO induces oxidative DNA damage, expression of the DNA repair enzyme MutS2, and mutations in cagY , demonstrating that DFMO directly affects genomic stability. Deletion of mutS2 abrogated the ability of DFMO to induce cagY rearrangements directly. In conclusion, DFMO-induced oxidative stress in H. pylori leads to genomic alterations and attenuates virulence., Competing Interests: The authors declare no conflict of interest.

Solute carrier family 7 member 2 (SLC7A2, also known as CAT2) is an inducible transporter of the semi-essential amino acid L-arginine (L-Arg), which has been implicated in wound repair. We have reported that both SLC7A2 expression and L-Arg availability are decreased in colonic tissues from inflammatory bowel disease patients and that mice lacking Slc7a2 exhibit a more severe disease course when exposed to dextran sulfate sodium (DSS) compared to wild-type (WT) mice. Here, we present evidence that SLC7A2 plays a role in modulating colon tumorigenesis in the azoxymethane (AOM)-DSS model of colitis-associated carcinogenesis (CAC). SLC7A2 was localized predominantly to colonic epithelial cells in WT mice. Utilizing the AOM-DSS model, Slc7a2 -/- mice had significantly increased tumor number, burden, and risk of high-grade dysplasia vs. WT mice. Tumors from Slc7a2 -/- mice exhibited significantly increased levels of the proinflammatory cytokines/chemokines IL-1β, CXCL1, CXCL5, IL-3, CXCL2, CCL3, and CCL4, but decreased levels of IL-4, CXCL9, and CXCL10 compared to tumors from WT mice. This was accompanied by a shift toward pro-tumorigenic M2 macrophage activation in Slc7a2-deficient mice, as marked by increased colonic CD11b + F4/80 + ARG1 + cells with no alteration in CD11b + F4/80 + NOS2 + cells by flow cytometry and immunofluorescence microscopy. The shift toward M2 macrophage activation was confirmed in bone marrow-derived macrophages from Slc7a2 -/- mice. In bone marrow chimeras between Slc7a2 -/- and WT mice, the recipient genotype drove the CAC phenotype, suggesting the importance of epithelial SLC7A2 in abrogating neoplastic risk. These data reveal that SLC7A2 has a significant role in the protection from CAC in the setting of chronic colitis, and suggest that the decreased SLC7A2 in inflammatory bowel disease (IBD) may contribute to CAC risk. Strategies to enhance L-Arg availability by supplementing L-Arg and/or increasing L-Arg uptake could represent a therapeutic approach in IBD to reduce the substantial long-term risk of colorectal carcinoma.

Helicobacter pylori infection induces a chronic gastric inflammation which can lead to gastric ulcers and cancer. The mucosal immune response to H. pylori is first initiated by the activation of gastric epithelial cells that respond to numerous bacterial factors, such as the cytotoxin-associated gene A or the lipopolysaccharide intermediate heptose-1,7-bisphosphate. The response of these cells is orchestrated by different receptors including the intracellular nucleotide-binding oligomerization domain-containing protein 1 or the extracellular epidermal growth factor receptor. This nonspecific response leads to recruitment and activation of various myeloid (macrophages and dendritic cells) and T cells (T helper-17 and mucosal-associated invariant T cells), which magnify and maintain inflammation. In this review, we summarize the major advances made in the past year regarding the induction, the regulation, and the role of the innate and adaptive immune responses to H. pylori infection. We also recapitulate efforts that have been made to develop efficient vaccine strategies., (© 2018 John Wiley & Sons Ltd.)

Blood vessel epicardial substance (BVES), or POPDC1, is a tight junction-associated transmembrane protein that modulates epithelial-to-mesenchymal transition (EMT) via junctional signaling pathways. There have been no in vivo studies investigating the role of BVES in colitis. We hypothesized that BVES is critical for maintaining colonic epithelial integrity. At baseline, Bves -/- mouse colons demonstrate increased crypt height, elevated proliferation, decreased apoptosis, altered intestinal lineage allocation, and dysregulation of tight junctions with functional deficits in permeability and altered intestinal immunity. Bves -/- mice inoculated with Citrobacter rodentium had greater colonic injury, increased colonic and mesenteric lymph node bacterial colonization, and altered immune responses after infection. We propose that increased bacterial colonization and translocation result in amplified immune responses and worsened injury. Similarly, dextran sodium sulfate (DSS) treatment resulted in greater histologic injury in Bves - /- mice. Two different human cell lines (Caco2 and HEK293Ts) co-cultured with enteropathogenic E. coli showed increased attaching/effacing lesions in the absence of BVES. Finally, BVES mRNA levels were reduced in human ulcerative colitis (UC) biopsy specimens. Collectively, these studies suggest that BVES plays a protective role both in ulcerative and infectious colitis and identify BVES as a critical protector of colonic mucosal integrity.

Ornithine decarboxylase (ODC) is the rate-limiting enzyme for polyamine biosynthesis and restricts M1 macrophage activation in gastrointestinal (GI) infections. However, the role of macrophage ODC in colonic epithelial-driven inflammation is unknown. Here, we investigate cell-specific effects of ODC in colitis and colitis-associated carcinogenesis (CAC). Human colonic macrophages expressed increased ODC levels in active ulcerative colitis and Crohn's disease, colitis-associated dysplasia, and CAC. Mice lacking Odc in myeloid cells ( Odc Δmye mice) that were treated with dextran sulfate sodium (DSS) exhibited improved survival, body weight, and colon length and reduced histologic injury versus control mice. In contrast, GI epithelial-specific Odc knockout had no effect on clinical parameters. Despite reduced histologic damage, colitis tissues of Odc Δmye mice had increased levels of multiple proinflammatory cytokines and chemokines and enhanced expression of M1, but not M2 markers. In the azoxymethane-DSS model of CAC, Odc Δmye mice had reduced tumor number, burden, and high-grade dysplasia. Tumors from Odc Δmye mice had increased M1, but not M2 macrophages. Increased levels of histone 3, lysine 9 acetylation, a marker of open chromatin, were manifest in tumor macrophages of Odc Δmye mice, consistent with our findings that macrophage ODC affects histone modifications that upregulate M1 gene transcription during GI infections. These findings support the concept that macrophage ODC augments epithelial injury-associated colitis and CAC by impairing the M1 responses that stimulate epithelial repair, antimicrobial defense, and antitumoral immunity. They also suggest that macrophage ODC is an important target for colon cancer chemoprevention. Significance: Ornithine decarboxylase contributes to the pathogenesis of colitis and associated carcinogenesis by impairing M1 macrophage responses needed for antitumoral immunity; targeting ODC in macrophages may represent a new strategy for chemoprevention. Cancer Res; 78(15); 4303-15. ©2018 AACR ., (©2018 American Association for Cancer Research.)

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 ( Efgr Δ epi 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 Egfr Δ epi versus Egfr fl/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. pylo ri-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 Efgr Δ epi versus Egfr fl/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., Competing Interests: Competing interests: None declared., (Published by the BMJ Publishing Group Limited. For permission to use (where not already granted under a licence) please go to http://www.bmj.com/company/products-services/rights-and-licensing/.)

Polyamines have been implicated in numerous biological processes, including inflammation and carcinogenesis. Homeostatic regulation leads to interconversion of the polyamines putrescine and the downstream metabolites spermidine and spermine. The enzyme spermine oxidase (SMOX), which back-converts spermine to spermidine, contributes to regulation of polyamine levels, but can also have other effects. We have implicated SMOX in gastric inflammation and carcinogenesis due to infection by the pathogen Helicobacter pylori . In addition, we reported that SMOX can be upregulated in humans with inflammatory bowel disease. Herein, we utilized Smox -deficient mice to examine the role of SMOX in two murine colitis models, Citrobacter rodentium infection and dextran sulfate sodium (DSS)-induced epithelial injury. In C. rodentium -infected wild-type (WT) mice, there were marked increases in colon weight/length and histologic injury, with mucosal hyperplasia and inflammatory cell infiltration; these changes were ameliorated in Smox -/- mice. In contrast, with DSS, Smox -/- mice exhibited substantial mortality, and increased body weight loss, colon weight/length, and histologic damage. In C. rodentium -infected WT mice, there were increased colonic levels of the chemokines CCL2, CCL3, CCL4, CXCL1, CXCL2, and CXCL10, and the cytokines IL-6, TNF-α, CSF3, IFN-γ, and IL-17; each were downregulated in Smox -/- mice. In DSS colitis, increased levels of IL-6, CSF3, and IL-17 were further increased in Smox -/- mice. In both models, putrescine and spermidine were increased in WT mice; in Smox -/- mice, the main effect was decreased spermidine and spermidine/spermine ratio. With C. rodentium , polyamine levels correlated with histologic injury, while with DSS, spermidine was inversely correlated with injury. Our studies indicate that SMOX has immunomodulatory effects in experimental colitis via polyamine flux. Thus, SMOX contributes to the immunopathogenesis of C. rodentium infection, but is protective in DSS colitis, indicating the divergent effects of spermidine.

Arginase activity induction in macrophages is an escape mechanism developed by parasites to cope with the host's immune defense and benefit from increased host-derived growth factor production. We report that arginase expression and activity were induced in macrophages during mouse infection by Trypanosoma musculi , a natural parasite of this host. This induction was reproduced in vitro by excreted/secreted factors of the parasite. A mAb directed to Tb KHC1, an orphan kinesin H chain from Trypanosoma brucei , inhibited T. musculi excreted/secreted factor-mediated arginase induction. Anti- Tb KHC1 Ab also inhibited T. musculi growth, both in vitro and in vivo. Induction of arginase activity and parasite growth involved C-type lectin receptors, because mannose injection decreased arginase activity induction and parasite load in vitro and in vivo. Accordingly, the parasite load was reduced in mice lacking mannose receptor C-type 1. The T. musculi KHC1 homolog showed high similarity with Tb KHC1. Bioinformatics analysis revealed the presence of homologs of this gene in other trypanosomes, including pathogens for humans and animals. Host metabolism dysregulation represents an effective parasite mechanism to hamper the host immune response and modify host molecule production to favor parasite invasion and growth. Thus, this orphan kinesin plays an important role in promoting trypanosome infection, and its neutralization or the lock of its partner host molecules offers promising approaches to increasing resistance to infection and new developments in vaccination against trypanosomiasis., (Copyright © 2017 by The American Association of Immunologists, Inc.)

Epidermal growth factor receptor (EGFR) signaling is a known mediator of colorectal carcinogenesis. Studies have focused on the role of EGFR signaling in epithelial cells, although the exact nature of the role of EGFR in colorectal carcinogenesis remains a topic of debate. Here, we present evidence that EGFR signaling in myeloid cells, specifically macrophages, is critical for colon tumorigenesis in the azoxymethane-dextran sodium sulfate (AOM-DSS) model of colitis-associated carcinogenesis (CAC). In a human tissue microarray, colonic macrophages demonstrated robust EGFR activation in the pre-cancerous stages of colitis and dysplasia. Utilizing the AOM-DSS model, mice with a myeloid-specific deletion of Egfr had significantly decreased tumor multiplicity and burden, protection from high-grade dysplasia and significantly reduced colitis. Intriguingly, mice with gastrointestinal epithelial cell-specific Egfr deletion demonstrated no differences in tumorigenesis in the AOM-DSS model. The alterations in tumorigenesis in myeloid-specific Egfr knockout mice were accompanied by decreased macrophage, neutrophil and T-cell infiltration. Pro-tumorigenic M2 macrophage activation was diminished in myeloid-specific Egfr-deficient mice, as marked by decreased Arg1 and Il10 mRNA expression and decreased interleukin (IL)-4, IL10 and IL-13 protein levels. Surprisingly, diminished M1 macrophage activation was also detectable, as marked by significantly reduced Nos2 and Il1b mRNA levels and decreased interferon (IFN)-γ, tumor necrosis factor (TNF)-α and IL-1β protein levels. The alterations in M1 and M2 macrophage activation were confirmed in bone marrow-derived macrophages from mice with the myeloid-specific Egfr knockout. The combined effect of restrained M1 and M2 macrophage activation resulted in decreased production of pro-angiogenic factors, CXCL1 and vascular endothelial growth factor (VEGF), and reduced CD31 + blood vessels, which likely contributed to protection from tumorigenesis. These data reveal that EGFR signaling in macrophages, but not in colonic epithelial cells, has a significant role in CAC. EGFR signaling in macrophages may prove to be an effective biomarker of CAC or target for chemoprevention in patients with inflammatory bowel disease.

Enterohemorrhagic Escherichia coli (EHEC) O157:H7 are human pathogens responsible for bloody diarrhea and renal failures. EHEC employ a type 3 secretion system to attach directly to the human colonic epithelium. This structure is encoded by the locus of enterocyte effacement (LEE) whose expression is regulated in response to specific nutrients. In this study, we show that the mucin-derived sugars N-acetylglucosamine (NAG) and N-acetylneuraminic acid (NANA) inhibit EHEC adhesion to epithelial cells through down-regulation of LEE expression. The effect of NAG and NANA is dependent on NagC, a transcriptional repressor of the NAG catabolism in E. coli . We show that NagC is an activator of the LEE1 operon and a critical regulator for the colonization of mice intestine by EHEC. Finally, we demonstrate that NAG and NANA as well as the metabolic activity of Bacteroides thetaiotaomicron affect the in vivo fitness of EHEC in a NagC-dependent manner. This study highlights the role of NagC in coordinating metabolism and LEE expression in EHEC and in promoting EHEC colonization in vivo .

Helicobacter pylori is a Gram-negative bacterium that specifically colonizes the gastric ecological niche. During the infectious process, which results in diseases ranging from chronic gastritis to gastric cancer, the host response is characterized by the activation of the innate immunity of gastric epithelial cells and macrophages. These cells thus produce effector molecules such as reactive oxygen species (ROS) to counteract the infection. The generation of ROS in response to H. pylori involves two canonical pathways: 1) the NADPH-dependent reduction of molecular oxygen to generate O 2 •- , which can dismute to generate ROS; and 2) the back-conversion of the polyamine spermine into spermidine through the enzyme spermine oxidase, leading to H 2 O 2 production. Although these products have the potential to affect the survival of bacteria, H. pylori has acquired numerous strategies to counteract their deleterious effects. Nonetheless, ROS-mediated oxidative DNA damage and mutations may participate in the adaptation of H. pylori to its ecological niche. Lastly, ROS have been shown to play a major role in the development of the inflammation and carcinogenesis. It is the purpose of this review to summarize the literature about the production of ROS during H. pylori infection and their role in this infectious gastric disease., (Published by Elsevier Inc.)

Macrophage activation is a critical step in host responses during bacterial infections. Ornithine decarboxylase (ODC), the rate-limiting enzyme in polyamine metabolism, has been well studied in epithelial cells and is known to have essential roles in many different cellular functions. However, its role in regulating macrophage function during bacterial infections is not well characterized. We demonstrate that macrophage-derived ODC is a critical regulator of M1 macrophage activation during both Helicobacter pylori and Citrobacter rodentium infection. Myeloid-specific Odc deletion significantly increased gastric and colonic inflammation, respectively, and enhanced M1 activation. Add-back of putrescine, the product of ODC, reversed the increased macrophage activation, indicating that ODC and putrescine are regulators of macrophage function. Odc-deficient macrophages had increased histone 3, lysine 4 (H3K4) monomethylation, and H3K9 acetylation, accompanied by decreased H3K9 di/trimethylation both in vivo and ex vivo in primary macrophages. These alterations in chromatin structure directly resulted in up-regulated gene transcription, especially M1 gene expression. Thus, ODC in macrophages tempers antimicrobial, M1 macrophage responses during bacterial infections through histone modifications and altered euchromatin formation, leading to the persistence and pathogenesis of these organisms., Competing Interests: The authors declare no conflict of interest.

The innate immune response is a critical hallmark of Helicobacter pylori infection. Epithelial and myeloid cells produce effectors, including the chemokine CXCL8, reactive oxygen species (ROS), and nitric oxide (NO), in response to bacterial components. Mechanistic and epidemiologic studies have emphasized that dysregulated and persistent release of these products leads to the development of chronic inflammation and to the molecular and cellular events related to carcinogenesis. Moreover, investigations in H. pylori-infected patients about polymorphisms of the genes encoding CXCL8 and inducible NO synthase, and epigenetic control of the ROS-producing enzyme spermine oxidase, have further proven that overproduction of these molecules impacts the severity of gastric diseases. Lastly, the critical effect of the crosstalk between the human host and the infecting bacterium in determining the severity of H. pylori-related diseases has been supported by phylogenetic analysis of the human population and their H. pylori isolates in geographic areas with varying clinical and pathologic outcomes of the infection.

The intestinal microbiota of patients with constipated-predominant irritable bowel syndrome (C-IBS) displays chronic dysbiosis. Our aim was to determine whether this microbial imbalance instigates perturbation of the host intestinal mucosal immune response, using a model of human microbiota-associated rats (HMAR) and dextran sulfate sodium (DSS)-induced experimental colitis. The analysis of the microbiota composition revealed a decrease of the relative abundance of Bacteroides, Roseburia-Eubacterium rectale and Bifidobacterium and an increase of Enterobacteriaceae, Desulfovibrio sp., and mainly Akkermansia muciniphila in C-IBS patients compared to healthy individuals. The bacterial diversity of the gut microbiota of healthy individuals or C-IBS patients was maintained in corresponding HMAR. Animals harboring a C-IBS microbiota had reduced DSS colitis with a decreased expression of pro-inflammatory cytokines from innate, Th1, and Th17 responses. The pre-treatment of conventional C57BL/6 mice or HMAR with A. muciniphila, but not with Escherichia coli, prior exposure to DSS also resulted in a reduction of colitis severity, highlighting that the anti-inflammatory effect of the gut microbiota of C-IBS patients is mediated, in part, by A. muciniphila. This work highlights a novel aspect of the crosstalk between the gut microbiota of C-IBS patients and host intestinal homeostasis.

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-1β, 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., Competing Interests: The authors have declared that no competing interests exist.

Shiga toxin-encoding Escherichia coli (STEC) regroup strains that carry genes encoding Shiga toxin (Stx). Among intestinal pathogenic E. coli, enterohaemorrhagic E. coli (EHEC) constitute the major subgroup of virulent STEC. EHEC cause serious human disease such as haemorrhagic colitis and haemolytic-uremic syndrome. While EHEC have evolved from enteropathogenic E. coli, hybrids with enteroaggregative E. coli have recently emerged. Of note, some enteroinvasive E. coli also belong to the STEC group. While the LEE (locus of enterocyte effacement) is a key and prominent molecular determinant in the pathogenicity, neither all EHEC nor STEC contain the LEE, suggesting that they possess additional virulence and colonisation factors. Currently, nine protein secretion systems have been described in diderm-lipopolysaccharide bacteria (archetypal Gram-negative) and can be involved in the secretion of extracellular effectors, cell-surface proteins or assembly of cell-surface organelles, such as flagella or pili. In this review, we focus on the secretome of STEC and related enteropathotypes, which are relevant to the colonisation of biotic and abiotic surfaces. Considering the wealth of potential protein trafficking mechanisms, the different combinations of colonisation factors and modulation of their expression is further emphasised with regard to the ecophysiology of STEC., (© FEMS 2016. All rights reserved. For permissions, please e-mail: journals.permissions@oup.com.)

Helicobacter pylori is a successful pathogen of the human stomach. Despite a vigorous immune response by the gastric mucosa, the bacterium survives in its ecological niche, thus favoring diseases ranging from chronic gastritis to adenocarcinoma. The current literature demonstrates that high-output of nitric oxide (NO) production by the inducible enzyme NO synthase-2 (NOS2) plays major functions in host defense against bacterial infections. However, pathogens have elaborated several strategies to counteract the deleterious effects of NO; this includes inhibition of host NO synthesis and transcriptional regulation in response to reactive nitrogen species, allowing the bacteria to face the nitrosative stress. Moreover, NO is also a critical mediator of inflammation and carcinogenesis. In this context, we review the recent findings on the expression of NOS2 in H. pylori-infected gastric tissues and epithelial cells, the role of NO in H. pylori-related diseases and H. pylori gene expression, and the mechanisms whereby H. pylori regulates NO synthesis by host cells., (Published by Elsevier Ltd.)

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., (Copyright © 2014 by The American Association of Immunologists, Inc.)

Expression of genes of the locus of enterocyte effacement (LEE) is essential for adherence of enterohemorrhagic Escherichia coli (EHEC) to intestinal epithelial cells. Gut factors that may modulate LEE gene expression may therefore influence the outcome of the infection. Because nitric oxide (NO) is a critical effector of the intestinal immune response that may induce transcriptional regulation in enterobacteria, we investigated its influence on LEE expression in EHEC O157:H7. We demonstrate that NO inhibits the expression of genes belonging to LEE1, LEE4, and LEE5 operons, and that the NO sensor nitrite-sensitive repressor (NsrR) is a positive regulator of these operons by interacting directly with the RNA polymerase complex. In the presence of NO, NsrR detaches from the LEE1/4/5 promoter regions and does not activate transcription. In parallel, two regulators of the acid resistance pathway, GadE and GadX, are induced by NO through an indirect NsrR-dependent mechanism. In this context, we show that the NO-dependent LEE1 down-regulation is due to absence of NsrR-mediated activation and to the repressor effect of GadX. Moreover, the inhibition of expression of LEE4 and LEE5 by NO is due to loss of NsrR-mediated activation, to LEE1 down-regulation and to GadE up-regulation. Lastly, we establish that chemical or cellular sources of NO inhibit the adherence of EHEC to human intestinal epithelial cells. These results highlight the critical effect of NsrR in the regulation of the LEE pathogenicity island and the potential role of NO in the limitation of colonization by EHEC.

Enterohemorrhagic Escherichia coli (EHEC) O157:H7 is a foodborne pathogen that resists the acidic gastric environment, colonizes the gut epithelium, and causes hemorrhagic colitis and hemolytic-uremic syndrome, especially in children. The genomic island OI-47 of E. coli O157:H7 contains a gene, z1528, encoding an EAL-domain protein potentially involved in c-di-GMP hydrolysis that is absent in non-pathogenic E. coli. This gene, designated vmpA, is co-transcribed with ycdT, which is present in non pathogenic E. coli and encodes a diguanylate cyclase involved in c-di-GMP synthesis. To test for vmpA function, we constructed a vmpA knockout mutant. We also overexpressed vmpA, purified the VmpA protein and assayed for its activity in vitro. We found that VmpA possesses c-di-GMP phosphodiesterase activity and that the vmpA mutation results in increased biofilm formation, and reduced swimming motility, which is consistent with the function determined in vitro. Unexpectedly, suppressor mutations arise frequently in the vmpA background suggesting that VmpA plays an important regulatory role in E. coli O157:H7. These findings represent an example of remarkable flexibility in the organization of c-di-GMP signaling pathways in closely related species., (Copyright © 2012 Elsevier B.V. All rights reserved.)

The cytotoxin-associated gene A protein (CagA) plays a pivotal role in the aetiology of Helicobacter pylori-associated gastric diseases. CagA is injected into the cytoplasm of host cells by a type IV secretion system, and is phosphorylated on tyrosine residues by the host enzyme c-Src. We previously reported that the enzyme haem oxygenase-1 (HO-1) inhibits IL-8 secretion by H. pylori-infected cells. However, the cellular mechanism by which HO-1 regulates the innate immune function of infected cells remains unknown. We now show that nitric oxide and haemin, two inducers of HO-1, decrease the level of phosphorylated CagA (p-CagA) in H. pylori-infected gastric epithelial cells and this is blocked by either pharmacological inhibition of HO-1 or siRNA knockdown of hmox-1. Moreover, forced expression of HO-1 by transfection of a plasmid expressing hmox-1 also results in a strong attenuation of CagA phosphorylation. This occurs through the inhibition of H. pylori-induced c-Src phosphorylation/activation by HO-1. Consequently, H. pylori-induced cytoskeletal rearrangements and activation of the pro-inflammatory response mediated by p-CagA are inhibited in HO-1-expressing cells. These data highlight a mechanism by which the innate immune response of the host can restrict the pathogenicity of H. pylori by attenuating CagA phosphorylation in gastric epithelial cells., (© 2012 Blackwell Publishing Ltd.)

L-arginine (L-Arg) is metabolized by nitric oxide synthase and arginase enzymes. The gastric pathogen Helicobacter pylori causes peptic ulcer disease and gastric cancer. We have shown that alterations in L-Arg availability and metabolism into polyamines contribute significantly to the dysregulation of the host immune response to this infection. Nitric oxide (NO) derived from inducible NO synthase (iNOS) can kill H. pylori. There are multiple mechanisms leading to failure of this process, including competition for L-Arg substrate by H. pylori arginase, and induction of host macrophage arginase II (Arg2) and ornithine decarboxylase (ODC). Generation of spermine by ODC inhibits iNOS translation and NO-mediated H. pylori killing. Expression of ODC is dependent on formation of a unique AP-1 complex, leading to upregulation of c-Myc as a transcriptional enhancer. Macrophage apoptosis is mediated by oxidation of spermine via the enzyme spermine oxidase (SMO) that generates hydrogen peroxide (H(2)O(2)), and thus oxidative stress-induced mitochondrial membrane polarization. Our studies have demonstrated that apoptosis occurs through a pERK → pc-Fos/c-Jun → c-Myc → ODC → SMO pathway. In gastric epithelial cells, activation of oxidative stress by H. pylori is dependent on SMO induction and results in both apoptosis and DNA damage, such that inhibition or knockdown of SMO markedly attenuates these events. In summary, L-Arg metabolism by the arginase-ODC pathway and the activation of SMO leads to H. pylori-induced DNA damage and immune dysregulation through polyamine-mediated oxidative stress and impairment of antimicrobial NO synthesis. Our studies indicate novel targets for therapeutic intervention in H. pylori-associated diseases, including gastritis, ulcer disease, and gastric cancer.

A strong cellular cross-talk exists between the pathogen Helicobacter pylori and high-output NO production. However, how NO and H. pylori interact to signal in gastric epithelial cells and modulate the innate immune response is unknown. We show that chemical or cellular sources of NO induce the anti-inflammatory effector heme oxygenase-1 (HO-1) in gastric epithelial cells through a pathway that requires NF-κB. However, H. pylori decreases NO-induced NF-κB activation, thereby inhibiting HO-1 expression. This inhibitory effect of H. pylori results from activation of the transcription factor heat shock factor-1 by the H. pylori virulence factor CagA and by the host signaling molecules ERK1/2 and JNK. Consistent with these findings, HO-1 is downregulated in gastric epithelial cells of patients infected with cagA(+) H. pylori but not in gastric epithelial cells of patients infected with cagA(-) H. pylori. Enhancement of HO-1 activity in infected cells or in H. pylori-infected mice inhibits chemokine generation and reduces inflammation. These data define a mechanism by which H. pylori favors its own pathogenesis by inhibiting HO-1 induction through the action of CagA.

Background & Aims: Helicobacter pylori-induced gastric carcinogenesis has been linked to the microbial oncoprotein cytotoxin-associated gene A (CagA). Spermine oxidase (SMO) metabolizes the polyamine spermine into spermidine and generates H(2)O(2), which causes apoptosis and DNA damage. We determined if pathogenic effects of CagA are attributable to SMO., Methods: Levels of SMO, apoptosis, and DNA damage (8-oxoguanosine) were measured in gastric epithelial cell lines infected with cagA(+) or cagA(-)H pylori strains, or transfected with a CagA expression plasmid, in the absence or presence of SMO small interfering RNA, or an SMO inhibitor. The role of CagA in induction of SMO and DNA damage was assessed in H pylori-infected gastritis tissues from humans, gerbils, and both wild-type and hypergastrinemic insulin-gastrin mice, using immunohistochemistry and flow cytometry., Results: cagA(+) strains or ectopic expression of CagA, but not cagA(-) strains, led to increased levels of SMO, apoptosis, and DNA damage in gastric epithelial cells, and knockdown or inhibition of SMO blocked apoptosis and DNA damage. There was increased SMO expression, apoptosis, and DNA damage in gastric tissues from humans infected with cagA(+), but not cagA(-) strains. In gerbils and mice, DNA damage was CagA-dependent and present in cells that expressed SMO. Gastric epithelial cells with DNA damage that were negative for markers of apoptosis accounted for 42%-69% of cells in gerbils and insulin-gastrin mice with dysplasia and carcinoma., Conclusions: By inducing SMO, H pylori CagA generates cells with oxidative DNA damage, and a subpopulation of these cells are resistant to apoptosis and thus at high risk for malignant transformation., (Copyright © 2011 AGA Institute. Published by Elsevier Inc. All rights reserved.)

Helicobacter pylori infection persists for the life of the host due to the failure of the immune response to eradicate the bacterium. Determining how H. pylori escapes the immune response in its gastric niche is clinically important. We have demonstrated in vitro that macrophage NO production can kill H. pylori, but induction of macrophage arginase II (Arg2) inhibits inducible NO synthase (iNOS) translation, causes apoptosis, and restricts bacterial killing. Using a chronic H. pylori infection model, we determined whether Arg2 impairs host defense in vivo. In C57BL/6 mice, expression of Arg2, but not arginase I, was abundant and localized to gastric macrophages. Arg2(-/-) mice had increased histologic gastritis and decreased bacterial colonization compared with wild-type (WT) mice. Increased gastritis scores correlated with decreased colonization in individual Arg2(-/-) mice but not in WT mice. When mice infected with H. pylori were compared, Arg2(-/-) mice had more gastric macrophages, more of these cells were iNOS(+), and these cells expressed higher levels of iNOS protein, as determined by flow cytometry and immunofluorescence microscopy. There was enhanced nitrotyrosine staining in infected Arg2(-/-) versus WT mice, indicating increased NO generation. Infected Arg2(-/-) mice exhibited decreased macrophage apoptosis, as well as enhanced IFN-γ, IL-17a, and IL-12p40 expression, and reduced IL-10 levels consistent with a more vigorous Th1/Th17 response. These studies demonstrate that Arg2 contributes to the immune evasion of H. pylori by limiting macrophage iNOS protein expression and NO production, mediating macrophage apoptosis, and restraining proinflammatory cytokine responses.