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.

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.

α-Difluoromethylornithine reduces gastric carcinogenesis by causing mutations in Helicobacter pylori cagY.

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.

Cardiac Extracellular Matrix Hydrogel Enriched with Polyethylene Glycol Presents Improved Gelation Time and Increased On-Target Site Retention of Extracellular Vesicles.

Stem-cell-derived extracellular vesicles (EVs) have demonstrated multiple beneficial effects in preclinical models of cardiac diseases. However, poor retention at the target site may limit their therapeutic efficacy. Cardiac extracellular matrix hydrogels (cECMH) seem promising as drug-delivery materials and could improve the retention of EVs, but may be limited by their long gelation time and soft mechanical properties. Our objective was to develop and characterize an optimized product combining cECMH, polyethylene glycol (PEG), and EVs (EVs-PEG-cECMH) in an attempt to overcome their individual limitations: long gelation time of the cECMH and poor retention of the EVs. The new combined product presented improved physicochemical properties (60% reduction in half gelation time, p < 0.001, and threefold increase in storage modulus, p < 0.01, vs. cECMH alone), while preserving injectability and biodegradability. It also maintained in vitro bioactivity of its individual components (55% reduction in cellular senescence vs. serum-free medium, p < 0.001, similar to EVs and cECMH alone) and increased on-site retention in vivo (fourfold increase vs. EVs alone, p < 0.05). In conclusion, the combination of EVs-PEG-cECMH is a potential multipronged product with improved gelation time and mechanical properties, increased on-site retention, and maintained bioactivity that, all together, may translate into boosted therapeutic efficacy.

Functional Properties of Helicobacter pylori VacA Toxin m1 and m2 Variants.

Helicobacter pylori colonizes the gastric mucosa and secretes a pore-forming toxin (VacA). Two main types of VacA, m1 and m2, can be distinguished by phylogenetic analysis. Type m1 forms of VacA have been extensively studied, but there has been relatively little study of m2 forms. In this study, we generated H. pylori strains producing chimeric proteins in which VacA m1 segments of a parental strain were replaced by corresponding m2 sequences. In comparison to the parental m1 VacA protein, a chimeric protein (designated m2/m1) containing m2 sequences in the N-terminal portion of the m region was less potent in causing vacuolation of HeLa cells, AGS gastric cells, and AZ-521 duodenal cells and had reduced capacity to cause membrane depolarization or death of AZ-521 cells. Consistent with the observed differences in activity, the chimeric m2/m1 VacA protein bound to cells at reduced levels compared to the binding levels of the parental m1 protein. The presence of two strain-specific insertions or deletions within or adjacent to the m region did not influence toxin activity. Experiments with human gastric organoids grown as monolayers indicated that m1 and m2/m1 forms of VacA had similar cell-vacuolating activities. Interestingly, both forms of VacA bound preferentially to the basolateral surface of organoid monolayers and caused increased cell vacuolation when interacting with the basolateral surface compared to the apical surface. These data provide insights into functional correlates of sequence variation in the VacA midregion (m region).

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.

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.

Induction of COX-2 expression by Helicobacter pylori is mediated by activation of epidermal growth factor receptor in gastric epithelial cells.

Chronic infection of the gastric mucosa by Helicobacter pylori is associated with an increased risk of developing gastric cancer; however, the vast majority of infected individuals never develop this disease. One H. pylori virulence factor that increases gastric cancer risk is the cag pathogenicity island, which encodes a bacterial type IV secretion system. Cyclooxygenase-2 (COX-2) expression is induced by proinflammatory stimuli, leading to increased prostaglandin E2 (PGE2) secretion by gastric epithelial cells. COX-2 expression is increased in gastric tissue from H. pylori-infected persons. H. pylori also activates the epidermal growth factor receptor (EGFR) in gastric epithelial cells. We now demonstrate that H. pylori-induced activation of COX-2 in gastric cells is dependent upon EGFR activation, and that a functional cag type IV secretion system and direct bacterial contact are necessary for full induction of COX-2 by gastric epithelial cells. PGE2 secretion is increased in cells infected with H. pylori, and this induction is dependent on a functional EGFR. Increased apoptosis in response to H. pylori occurs in cells treated with a COX-2 inhibitor, as well as COX-2-/- cells, indicating that COX-2 expression promotes cell survival. In vivo, COX-2 induction by H. pylori is significantly reduced in mice deficient for EGFR activation compared with wild-type mice with a fully functional receptor. Collectively, these findings indicate that aberrant activation of the EGFR-COX-2 axis may lower the threshold for carcinogenesis associated with chronic H. pylori infection.

Cystathionine γ-lyase exacerbates Helicobacter pylori immunopathogenesis by promoting macrophage metabolic remodeling and activation.

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.

Antiviral activity against Zika virus of a new formulation of curcumin in poly lactic-co-glycolic acid nanoparticles.

OBJECTIVES: In the search of an effective antiviral formulation, the natural product curcumin (CUR) was encapsulated into poly(lactic-co-glycolic acid) nanoparticles, a non-toxic bioresorbable and biocompatible copolymer. The resulting CUR containing particles (PLGA-CUR NPs) were characterized and analysed for antiviral activity against Zika virus (ZIKV) infection. METHODS: The PLGA-CUR NPs were characterized by Fourier transform infrared, differential scanning calorimetry, dynamic light scattering, scanning electron microscopy and thermogravimetric analysis and release profile. Cytotoxicity of PLGA-CUR and the antiviral activity against ZIKV were determined in Vero cells. The effect of PLGA-CUR NPs on viral RNA synthesis and protein expression was analysed by RT-qPCR and immunofluorescence staining, respectively. KEY FINDINGS: The PLGA-CUR NPs showed an appropriate in vitro drug release profile. Our studies of the antiviral activity of PLGA-CUR NPs and CUR against ZIKV by virus yield reduction as well as viral RNA synthesis and protein expression have shown that PLGA-CUR formulation is more effective than free CUR to inhibit ZIKV infection of Vero cells. CONCLUSIONS: Our results demonstrate for the first time the antiviral activity against ZIKV of PLGA nanoparticles charged with CUR, suggesting that PLGA-CUR NPs are promising candidates for a drug formulation against human pathogenic flaviviruses.

Role of Hcp, a type 6 secretion system effector, of Aeromonas hydrophila in modulating activation of host immune cells.

Recently, we reported that the type 6 secretion system (T6SS) of Aeromonas hydrophila SSU plays an important role in bacterial virulence in a mouse model, and immunization of animals with the T6SS effector haemolysin co-regulated protein (Hcp) protected them against lethal infections with wild-type bacteria. Additionally, we showed that the mutant bacteria deleted for the vasH gene within the T6SS gene cluster did not express the hcp gene, while the vasK mutant could express and translocate Hcp, but was unable to secrete it into the extracellular milieu. Both of these A. hydrophila SSU mutants were readily phagocytosed by murine macrophages, pointing to the possible role of the secreted form of Hcp in the evasion of the host innate immunity. By using the ΔvasH mutant of A. hydrophila, our in vitro data showed that the addition of exogenous recombinant Hcp (rHcp) reduced bacterial uptake by macrophages. These results were substantiated by increased bacterial virulence when rHcp was added along with the ΔvasH mutant in a septicaemic mouse model of infection. Analysis of the cytokine profiling in the intraperitoneal lavage as well as activation of host cells after 4 h of infection with the ΔvasH mutant supplemented with rHcp indicated that this T6SS effector inhibited production of pro-inflammatory cytokines and induced immunosuppressive cytokines, such as interleukin-10 and transforming growth factor-ß, which could circumvent macrophage activation and maturation. This mechanism of innate immune evasion by Hcp possibly inhibited the recruitment of cellular immune components, which allowed bacterial multiplication and dissemination in animals, thereby leading to their mortality.

Unraveling the mechanism of action of a new type III secretion system effector AexU from Aeromonas hydrophila.

We recently characterized a T3SS effector, AexU, from a diarrheal isolate SSU of Aeromonas hydrophila, which exhibited ADP-ribosyltransferase (ADPRT) activity. Here we provided evidence that AexU also possessed GTPase-activating protein (GAP) activity, which was mainly responsible for host cell apoptosis and disruption of actin filaments. Earlier, we showed that the DeltaaexU null mutant was attenuated in a mouse model, and we now demonstrated that while the parental A. hydrophila strain could be detected in the lung, liver, and spleen of infected mice, the DeltaaexU mutant was rapidly cleared from these organs resulting in increased survivability of animals. Further, AexU prevented phosphorylation of c-Jun, JNK and IkappaBalpha and inhibited IL-6 and IL-8 secretion from HeLa cells. Our data indicated that AexU operated by inhibiting NF-kappaB and inactivating Rho GTPases. Importantly, however, when the DeltaaexU null mutant was complemented with the mutated aexU gene devoid of ADPRT and GAP activities, a higher mortality rate in mice with concomitant increase in the production of pro-inflammatory cytokines/chemokines was noted. These data indicated that either such a mutated AexU is a potent inducer of them or that AexU possesses yet another unknown activity that is modulated by ADPRT and GAP activities and results in this aberrant cytokine/chemokine production responsible for increased animal death.

Hypusination Orchestrates the Antimicrobial Response of Macrophages.

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 (EIF5AHyp) 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. EIF5AHyp 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 EIF5AHyp 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.

Spermine oxidase mediates Helicobacter pylori-induced gastric inflammation, DNA damage, and carcinogenic signaling.

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 H2O2, 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 ß-catenin activation in H. pylori-infected Smox-/- mice or gastric organoids, compared to infected wild-type animals or gastroids. The link between SMOX and ß-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 ß-catenin signaling.

N-acylhomoserine lactones involved in quorum sensing control the type VI secretion system, biofilm formation, protease production, and in vivo virulence in a clinical isolate of Aeromonas hydrophila.

In this study, we delineated the role of N-acylhomoserine lactone(s) (AHLs)-mediated quorum sensing (QS) in the virulence of diarrhoeal isolate SSU of Aeromonas hydrophila by generating a double knockout Delta ahyRI mutant. Protease production was substantially reduced in the Delta ahyRI mutant when compared with that in the wild-type (WT) strain. Importantly, based on Western blot analysis, the Delta ahyRI mutant was unable to secrete type VI secretion system (T6SS)-associated effectors, namely haemolysin coregulated protein and the valine-glycine repeat family of proteins, while significant levels of these effectors were detected in the culture supernatant of the WT A. hydrophila. In contrast, the production and translocation of the type III secretion system (T3SS) effector AexU in human colonic epithelial cells were not affected when the ahyRI genes were deleted. Solid surface-associated biofilm formation was significantly reduced in the Delta ahyRI mutant when compared with that in the WT strain, as determined by a crystal violet staining assay. Scanning electron microscopic observations revealed that the Delta ahyRI mutant was also defective in the formation of structured biofilm, as it was less filamentous and produced a distinct exopolysaccharide on its surface when compared with the structured biofilm produced by the WT strain. These effects of AhyRI could be complemented either by expressing the ahyRI genes in trans or by the exogeneous addition of AHLs to the Delta ahyRI/ahyR(+) complemented strain. In a mouse lethality experiment, 50 % attenuation was observed when we deleted the ahyRI genes from the parental strain of A. hydrophila. Together, our data suggest that AHL-mediated QS modulates the virulence of A. hydrophila SSU by regulating the T6SS, metalloprotease production and biofilm formation.

Alteration in the activation state of new inflammation-associated targets by phospholipase A2-activating protein (PLAA).

Phospholipase A(2) (PLA(2))-activating protein (PLAA) is a novel signaling molecule that regulates the production of prostaglandins (PGE(2)) and tumor necrosis factor (TNF)-alpha. To characterize the function of native PLAA in situ, we generated HeLa (Tet-off) cells overexpressing plaa (plaa(high)) and control (plaa(low)) cells, with the plaa gene in opposite orientation in the latter construct. The plaa(high) cells produced significantly more PGE(2) and interleukin (IL)-6 compared to plaa(low) cells in response to TNF-alpha. There was an increased activation and/or expression of cytosolic PLA(2), cyclooxgenase-2, and NF-kappaB after induction of plaa(high) cells with TNF-alpha compared to the respective plaa(low) cells. Microarray analysis of plaa(high) cells followed by functional assays revealed increased production of proinflammatory cytokine IL-32 and a decrease in the production of annexin A4 and clusterin compared to plaa(low) cells. We demonstrated the role of annexin A4 as an inhibitor of PLA(2) and showed that addition of exogeneous clusterin limited the production of PGE(2) from plaa(high) cells. To understand regulation of plaa gene expression, we used a luciferase reporter system in HeLa cells and identified one stimulatory element, with Sp1 binding sites, and one inhibitory element, in exon 1 of the plaa gene. By using decoy DNA oligonucleotides to Sp1 and competitive binding assays, we showed that Sp1 maintains basal expression of the plaa gene and binds to the above-mentioned stimulatory element. We demonstrated for the first time that the induction of native PLAA by TNF-alpha can perpetuate inflammation by enhancing activation of PLA(2) and NF-kappaB.

Nod1 Imprints Inflammatory and Carcinogenic Responses toward the Gastric Pathogen Helicobacter pylori.

Helicobacter pylori (H. pylori) is the strongest known risk for gastric cancer. The H. pylori cag type IV secretion system is an oncogenic locus that translocates peptidoglycan into host cells, where it is recognized by NOD1, an innate immune receptor. Beyond this, the role of NOD1 in H. pylori-induced cancer remains undefined. To address this knowledge gap, we infected two genetic models of Nod1 deficiency with the H. pylori cag + strain PMSS1: C57BL/6 mice, which rarely develop cancer, and INS-GAS FVB/N mice, which commonly develop cancer. Infected C57BL/6 Nod1-/- and INS-GAS Nod1-/- mice acutely developed more severe gastritis, and INS-GAS Nod1-/- mice developed gastric dysplasia more frequently compared with Nod1+/+ mice. Because Nod1 genotype status did not alter microbial phenotypes of in vivo-adapted H. pylori, we investigated host immunologic responses. H. pylori infection of Nod1-/- mice led to significantly increased gastric mucosal levels of Th1, Th17, and Th2 cytokines compared with Nod1 wild-type (WT) mice. To define the role of specific innate immune cells, we quantified cytokine secretion from H. pylori-infected primary gastric organoids generated from WT or Nod1-/- mice that were cocultured with or without WT or Nod1-/- macrophages. Infection increased cytokine production from gastric epithelial cells and macrophages and elevations were significantly increased with Nod1 deficiency. Furthermore, H. pylori infection altered the polarization status of Nod1-/- macrophages compared with Nod1+/+ macrophages. Collectively, these studies demonstrate that loss of Nod1 augments inflammatory and injury responses to H. pylori. Nod1 may exert its restrictive role by altering macrophage polarization, leading to immune evasion and microbial persistence. SIGNIFICANCE: These findings suggest that manipulation of NOD1 may represent a novel strategy to prevent or treat pathologic outcomes induced by H. pylori infection.

Bacterial Pathogens Hijack the Innate Immune Response by Activation of the Reverse Transsulfuration Pathway.

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.

Distinct Immunomodulatory Effects of Spermine Oxidase in Colitis Induced by Epithelial Injury or Infection.

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.