Cryptosporidium parvum increases intestinal permeability through interaction with epithelial cells and IL-1ß and TNFα released by inflammatory monocytes.

Intestinal epithelial cells form a single layer separating the intestinal lumen containing nutriments and microbiota from the underlying sterile tissue and therefore play a key role in maintaining homeostasis. We investigated the factors contributing to the alteration of the epithelial barrier function during Cryptosporidium parvum infection. Infected polarized epithelial cell monolayers exhibit a drop in transepithelial resistance associated with a delocalization of E-cadherin and ß-catenin from their intercellular area of contact, the adherens junction complex. In neonatal mice infected by C. parvum, the increased permeability is correlated with parasite development and with an important recruitment of Ly6c+ inflammatory monocytes to the subepithelial space. TNFα and IL-1ß produced by inflammatory monocytes play a key role in the loss of barrier function. Our findings demonstrate for the first time that both the parasite and inflammatory monocytes contribute to the loss of intestinal barrier function during cryptosporidiosis.

Reduced parasite motility and micronemal protein secretion by a p38 MAPK inhibitor leads to a severe impairment of cell invasion by the apicomplexan parasite Eimeria tenella.

E. tenella infection is associated with a severe intestinal disease leading to high economic losses in poultry industry. Mitogen activated protein kinases (MAPKs) are implicated in early response to infection and are divided in three pathways: p38, extracellular signal-regulated protein kinase (ERK) and c-Jun N-terminal kinase (JNK). Our objective was to determine the importance of these kinases on cell invasion by E. tenella. We evaluated the effect of specific inhibitors (ERK: PD98059, JNKII: SP600125, p38 MAPK: SB203580) on the invasion of epithelial cells. Incubation of SP600125 and SB203580 with epithelial cells and parasites significantly inhibited cell invasion with the highest degree of inhibition (90%) for SB203580. Silencing of the host p38α MAPK expression by siRNA led to only 20% decrease in cell invasion. In addition, when mammalian epithelial cells were pre-treated with SB203580, and washed prior infection, a 30% decrease in cell invasion was observed. This decrease was overcome when a p38 MAPK activator, anisomycin was added during infection. This suggests an active but limited role of the host p38 MAPK in this process. We next determined whether SB203580 has a direct effect on the parasite. Indeed, parasite motility and secretion of micronemal proteins (EtMIC1, 2, 3 and 5) that are involved in cell invasion were both decreased in the presence of the inhibitor. After chasing the inhibitor, parasite motility and secretion of micronemal proteins were restored and subsequently cell invasion. SB203580 inhibits cell invasion by acting partly on the host cell and mainly on the parasite.

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

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

Human and Helicobacter pylori coevolution shapes the risk of gastric disease.

Helicobacter pylori is the principal cause of gastric cancer, the second leading cause of cancer mortality worldwide. However, H. pylori prevalence generally does not predict cancer incidence. To determine whether coevolution between host and pathogen influences disease risk, we examined the association between the severity of gastric lesions and patterns of genomic variation in matched human and H. pylori samples. Patients were recruited from two geographically distinct Colombian populations with significantly different incidences of gastric cancer, but virtually identical prevalence of H. pylori infection. All H. pylori isolates contained the genetic signatures of multiple ancestries, with an ancestral African cluster predominating in a low-risk, coastal population and a European cluster in a high-risk, mountain population. The human ancestry of the biopsied individuals also varied with geography, with mostly African ancestry in the coastal region (58%), and mostly Amerindian ancestry in the mountain region (67%). The interaction between the host and pathogen ancestries completely accounted for the difference in the severity of gastric lesions in the two regions of Colombia. In particular, African H. pylori ancestry was relatively benign in humans of African ancestry but was deleterious in individuals with substantial Amerindian ancestry. Thus, coevolution likely modulated disease risk, and the disruption of coevolved human and H. pylori genomes can explain the high incidence of gastric disease in the mountain population.

Virulence of infecting Helicobacter pylori strains and intensity of mononuclear cell infiltration are associated with levels of DNA hypermethylation in gastric mucosae.

DNA methylation changes are known to occur in gastric cancers and in premalignant lesions of the gastric mucosae. In order to examine variables associated with methylation levels, we quantitatively evaluated DNA methylation in tumors, non-tumor gastric mucosae, and in gastric biopsies at promoters of 5 genes with methylation alterations that discriminate gastric cancers from non-tumor epithelia (EN1, PCDH10, RSPO2, ZIC1, and ZNF610). Among Colombian subjects at high and low risk for gastric cancer, biopsies from subjects from the high-risk region had significantly higher levels of methylation at these 5 genes than samples from subjects in the low risk region (p ≤ 0.003). When results were stratified by Helicobacter pylori infection status, infection with a cagA positive, vacA s1m1 strain was significantly associated with highest methylation levels, compared with other strains (p = 0.024 to 0.001). More severe gastric inflammation and more advanced precancerous lesions were also associated with higher levels of DNA methylation (p ≤ 0.001). In a multivariate model, location of residence of the subject and the presence of cagA and vacA s1m1 in the H. pylori strain were independent variables associated with higher methylation in all 5 genes. High levels of mononuclear cell infiltration were significantly related to methylation in PCDH10, RSPO2, and ZIC1 genes. These results indicate that for these genes, levels of methylation in precancerous lesions are related to H. pylori virulence, geographic region and measures of chronic inflammation. These genes seem predisposed to sustain significant quantitative changes in DNA methylation at early stages of the gastric precancerous process.

Chronic inflammation and oxidative stress: the smoking gun for Helicobacter pylori-induced gastric cancer?

Helicobacter pylori is the leading risk factor associated with gastric carcinogenesis. H. pylori leads to chronic inflammation because of the failure of the host to eradicate the infection. Chronic inflammation leads to oxidative stress, deriving from immune cells and from within gastric epithelial cells. This is a main contributor to DNA damage, apoptosis and neoplastic transformation. Both pathogen and host factors directly contribute to oxidative stress, including H. pylori virulence factors, and pathways involving DNA damage and repair, polyamine synthesis and metabolism, and oxidative stress response. Our laboratory has recently uncovered a mechanism by which polyamine oxidation by spermine oxidase causes H 2O 2 release, DNA damage and apoptosis. Our studies indicate novel targets for therapeutic intervention and risk assessment in H. pylori-induced gastric cancer. More studies addressing the many potential contributors to oxidative stress, chronic inflammation, and gastric carcinogenesis are essential for development of therapeutics and identification of gastric cancer biomarkers.

Haem oxygenase-1 inhibits phosphorylation of the Helicobacter pylori oncoprotein CagA in gastric epithelial cells.

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.

Spermine oxidase, a polyamine catabolic enzyme that links Helicobacter pylori CagA and gastric cancer risk.

We have recently reported that Helicobacter pylori strains expressing the virulence factor cytotoxin-associated gene A (CagA) stimulate increased levels of spermine oxidase (SMO) in gastric epithelial cells, while cagA⁻ strains did not. SMO catabolizes the polyamine spermine and produces H2O2 that results in both apoptosis and DNA damage. Exogenous overexpression of CagA confirmed these findings, and knockdown or inhibition of SMO blocked CagA-mediated apoptosis and DNA damage. The strong association of SMO, apoptosis, and DNA damage was also demonstrated in humans infected with cagA⁺, but not cagA⁻ strains. In infected gerbils and mice, DNA damage was CagA-dependent and only present in epithelial cells that expressed SMO. We also discovered SMO (high) gastric epithelial cells from infected animals with dysplasia that are resistant to apoptosis despite high levels of DNA damage. Inhibition of polyamine synthesis or SMO could abrogate the development of this cell population that may represent precursors for neoplastic transformation.

Arginine and polyamines in Helicobacter pylori-induced immune dysregulation and gastric carcinogenesis.

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.

Cationic amino acid transporter 2 enhances innate immunity during Helicobacter pylori infection.

Once acquired, Helicobacter pylori infection is lifelong due to an inadequate innate and adaptive immune response. Our previous studies indicate that interactions among the various pathways of arginine metabolism in the host are critical determinants of outcomes following infection. Cationic amino acid transporter 2 (CAT2) is essential for transport of L-arginine (L-Arg) into monocytic immune cells during H. pylori infection. Once within the cell, this amino acid is utilized by opposing pathways that lead to elaboration of either bactericidal nitric oxide (NO) produced from inducible NO synthase (iNOS), or hydrogen peroxide, which causes macrophage apoptosis, via arginase and the polyamine pathway. Because of its central role in controlling L-Arg availability in macrophages, we investigated the importance of CAT2 in vivo during H. pylori infection. CAT2(-/-) mice infected for 4 months exhibited decreased gastritis and increased levels of colonization compared to wild type mice. We observed suppression of gastric macrophage levels, macrophage expression of iNOS, dendritic cell activation, and expression of granulocyte-colony stimulating factor in CAT2(-/-) mice suggesting that CAT2 is involved in enhancing the innate immune response. In addition, cytokine expression in CAT2(-/-) mice was altered from an antimicrobial Th1 response to a Th2 response, indicating that the transporter has downstream effects on adaptive immunity as well. These findings demonstrate that CAT2 is an important regulator of the immune response during H. pylori infection.

Disruption of nitric oxide signaling by Helicobacter pylori results in enhanced inflammation by inhibition of heme oxygenase-1.

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.

Spermine oxidase mediates the gastric cancer risk associated with Helicobacter pylori 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.

Phylogeographic origin of Helicobacter pylori is a determinant of gastric cancer risk.

BACKGROUND AND AIMS: Helicobacter pylori colonises the stomach in half of all humans, and is the principal cause of gastric cancer, the second leading cause of cancer death worldwide. While gastric cancer rates correlate with H pylori prevalence in some areas, there are regions where infection is nearly universal, but rates of gastric cancer are low. In the case of Colombia, there is a 25-fold increase in gastric cancer rate in the Andean mountain (high risk) region compared to the coastal (low risk) region, despite similarly high (∼90%) prevalence of H pylori in the two locations. Our aim was to investigate the ancestral origin of H pylori strains isolated from subjects in these high- and low-risk regions and to determine whether this is a predictive determinant of precancerous lesions. METHODS: Multi-locus sequence typing was used to investigate phylogeographic origins of infecting H pylori strains isolated from subjects in the Pacific coast and Andes Mountains in the state of Nariño, Colombia. We analysed 64 subjects infected with cagA+ vacA s1m1 strains. Gastric biopsy slides from each individual were scored for histological lesions and evaluated for DNA damage by immunohistochemistry. RESULTS: We show that strains from the high-risk region were all of European phylogeographic origin, whereas those from the low risk region were of either European (34%) or African origin (66%). European strain origin was strongly predictive of increased premalignant histological lesions and epithelial DNA damage, even in the low-risk region; African strain origin was associated with reduced severity of these parameters. CONCLUSION: The phylogeographic origin of H pylori strains provides an explanation for geographic differences in cancer risk deriving from this infection.

Difluoromethylornithine is a novel inhibitor of Helicobacter pylori growth, CagA translocation, and interleukin-8 induction.

Helicobacter pylori infects half the world's population, and carriage is lifelong without antibiotic therapy. Current regimens prescribed to prevent infection-associated diseases such as gastroduodenal ulcers and gastric cancer can be thwarted by antibiotic resistance. We reported that administration of 1% D,L-α-difluoromethylornithine (DFMO) to mice infected with H. pylori reduces gastritis and colonization, which we attributed to enhanced host immune response due to inhibition of macrophage ornithine decarboxylase (ODC), the rate-limiting enzyme in polyamine biosynthesis. Although no ODC has been identified in any H. pylori genome, we sought to determine if DFMO has direct effects on the bacterium. We found that DFMO significantly reduced the growth rate of H. pylori in a polyamine-independent manner. Two other gram-negative pathogens possessing ODC, Escherichia coli and Citrobacter rodentium, were resistant to the DFMO effect. The effect of DFMO on H. pylori required continuous exposure to the drug and was reversible when removed, with recovery of growth rate in vitro and the ability to colonize mice. H. pylori exposed to DFMO were significantly shorter in length than those untreated and they contained greater internal levels of ATP, suggesting severe effects on bacterial metabolism. DFMO inhibited expression of the H. pylori virulence factor cytotoxin associated gene A, and its translocation and phosphorylation in gastric epithelial cells, which was associated with a reduction in interleukin-8 expression. These findings suggest that DFMO has effects on H. pylori that may contribute to its effectiveness in reducing gastritis and colonization and may be a useful addition to anti-H. pylori therapies.

Immune evasion by Helicobacter pylori is mediated by induction of macrophage arginase II.

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.

Polyamines Impair Immunity to Helicobacter pylori by Inhibiting L-Arginine Uptake Required for Nitric Oxide Production.

BACKGROUND & AIMS: Helicobacter pylori-induced immune responses fail to eradicate the bacterium. Nitric oxide (NO) can kill H pylori. However, translation of inducible NO synthase (iNOS) and NO generation by H pylori-stimulated macrophages is inhibited by the polyamine spermine derived from ornithine decarboxylase (ODC), and is dependent on availability of the iNOS substrate L-arginine (L-Arg). We determined if spermine inhibits iNOS-mediated immunity by reducing L-Arg uptake into macrophages. METHODS: Levels of the inducible cationic amino acid transporter (CAT)2, ODC, and iNOS were measured in macrophages and H pylori gastritis tissues. L-Arg uptake, iNOS expression, and NO levels were assessed in cells with small interfering RNA knockdown of CAT2 or ODC, and in gastric macrophages. The ODC inhibitor, α-difluoromethylornithine, was administered to H pylori-infected mice for 4 months after inoculation. RESULTS: H pylori induced CAT2 and uptake of L-Arg in RAW 264.7 or primary macrophages. Addition of spermine or knockdown of CAT2 inhibited L-Arg uptake, NO production, and iNOS protein levels, whereas knockdown of ODC had the opposite effect. CAT2 and ODC were increased in mouse and human H pylori gastritis tissues and localized to macrophages. Gastric macrophages from H pylori-infected mice showed increased ODC expression, and attenuated iNOS and NO levels upon ex vivo H pylori stimulation versus cells from uninfected mice. α-Difluoromethylornithine treatment of infected mice restored L-Arg uptake, iNOS protein expression, and NO production in gastric macrophages, and significantly reduced both H pylori colonization levels and gastritis severity. CONCLUSIONS: Up-regulation of ODC in gastric macrophages impairs host defense against H pylori by suppressing iNOS-derived NO production.

Helicobacter pylori induces ERK-dependent formation of a phospho-c-Fos c-Jun activator protein-1 complex that causes apoptosis in macrophages.

Macrophages are essential components of innate immunity, and apoptosis of these cells impairs mucosal defense to microbes. Helicobacter pylori is a gastric pathogen that infects half of the world population and causes peptic ulcer disease and gastric cancer. The host inflammatory response fails to eradicate the organism. We have reported that H. pylori induces apoptosis of macrophages by generation of polyamines from ornithine decarboxylase (ODC), which is dependent on c-Myc as a transcriptional enhancer. We have now demonstrated that expression of c-Myc requires phosphorylation and nuclear translocation of ERK, which results in phosphorylation of c-Fos and formation of a specific activator protein (AP)-1 complex. Electromobility shift assay and immunoprecipitation revealed a previously unrecognized complex of phospho-c-Fos (pc-Fos) and c-Jun in the nucleus. Fluorescence resonance energy transfer demonstrated the interaction of pc-Fos and c-Jun. The capacity of this AP-1 complex to bind to putative AP-1 sequences was demonstrated by oligonucleotide pulldown and fluorescence polarization. Binding of the pc-Fos.c-Jun complex to the c-Myc promoter was demonstrated by chromatin immunoprecipitation. A dominant-negative c-Fos inhibited H. pylori-induced expression of c-Myc and ODC and apoptosis. H. pylori infection of mice induced a rapid infiltration of macrophages into the stomach. Concomitant apoptosis depleted these cells, and this was associated with formation of a pc-Fos.c-Jun complex. Treatment of mice with an inhibitor of ERK phosphorylation attenuated phosphorylation of c-Fos, expression of ODC, and apoptosis in gastric macrophages. A unique AP-1 complex in gastric macrophages contributes to the immune escape of H. pylori.

Arginase II restricts host defense to Helicobacter pylori by attenuating inducible nitric oxide synthase translation in macrophages.

Helicobacter pylori infection of the stomach causes peptic ulcer disease and gastric cancer. Despite eliciting a vigorous immune response, the bacterium persists for the life of the host. An important antimicrobial mechanism is the production of NO derived from inducible NO synthase (iNOS). We have reported that macrophages can kill H. pylori in vitro by an NO-dependent mechanism, but supraphysiologic levels of the iNOS substrate l-arginine are required. Because H. pylori induces arginase activity in macrophages, we determined if this restricts NO generation by reducing l-arginine availability. Inhibition of arginase with S-(2-boronoethyl)-l-cysteine (BEC) significantly enhanced NO generation in H. pylori-stimulated RAW 264.7 macrophages by enhancing iNOS protein translation but not iNOS mRNA levels. This effect resulted in increased killing of H. pylori that was attenuated with an NO scavenger. In contrast, inhibition of arginase in macrophages activated by the colitis-inducing bacterium Citrobacter rodentium increased NO without affecting iNOS levels. H. pylori upregulated levels of arginase II (Arg2) mRNA and protein, which localized to mitochondria, whereas arginase I was not induced. Increased iNOS protein and NO levels were also demonstrated by small interfering RNA knockdown of Arg2 and in peritoneal macrophages from C57BL/6 Arg2(-/-) mice. In H. pylori-infected mice, treatment with BEC or deletion of Arg2 increased iNOS protein levels and NO generation in gastric macrophages, but treatment of Arg2(-/-) mice with BEC had no additional effect. These studies implicate Arg2 in the immune evasion of H. pylori by causing intracellular depletion of l-arginine and thus reduction of NO-dependent bactericidal activity.

Human microbiota-secreted factors inhibit shiga toxin synthesis by enterohemorrhagic Escherichia coli O157:H7.

Escherichia coli O157:H7 is a food-borne pathogen causing hemorrhagic colitis and hemolytic-uremic syndrome, especially in children. The main virulence factor responsible for the more serious disease is the Shiga toxin 2 (Stx2), which is released in the gut after oral ingestion of the organism. Although it is accepted that the amount of Stx2 produced by E. coli O157:H7 in the gut is critical for the development of disease, the eukaryotic or prokaryotic gut factors that modulate Stx2 synthesis are largely unknown. In this study, we examined the influence of prokaryotic molecules released by a complex human microbiota on Stx2 synthesis by E. coli O157:H7. Stx2 synthesis was assessed after growth of E. coli O157:H7 in cecal contents of gnotobiotic rats colonized with human microbiota or in conditioned medium having supported the growth of complex human microbiota. Extracellular prokaryotic molecules produced by the commensal microbiota repress stx(2) mRNA expression and Stx2 production by inhibiting the spontaneous and induced lytic cycle mediated by RecA. These molecules, with a molecular mass of below 3 kDa, are produced in part by Bacteroides thetaiotaomicron, a predominant species of the normal human intestinal microbiota. The microbiota-induced stx(2) repression is independent of the known quorum-sensing pathways described in E. coli O157:H7 involving SdiA, QseA, QseC, or autoinducer 3. Our findings demonstrate for the first time the regulatory activity of a soluble factor produced by the complex human digestive microbiota on a bacterial virulence factor in a physiologically relevant context.

Heme oxygenase-1 is a critical regulator of nitric oxide production in enterohemorrhagic Escherichia coli-infected human enterocytes.

Enterohemorrhagic Escherichia coli (EHEC) are the causative agent of hemolytic-uremic syndrome. In the first stage of the infection, EHEC interact with human enterocytes to modulate the innate immune response. Inducible NO synthase (iNOS)-derived NO is a critical mediator of the inflammatory response of the infected intestinal mucosa. We therefore aimed to analyze the role of EHEC on iNOS induction in human epithelial cell lines. In this regard, we show that EHEC down-regulate IFN-gamma-induced iNOS mRNA expression and NO production in Hct-8, Caco-2, and T84 cells. This inhibitory effect occurs through the decrease of STAT-1 activation. In parallel, we demonstrate that EHEC stimulate the rapid inducible expression of the gene hmox-1 that encodes for the enzyme heme oxygenase-1 (HO-1). Knock-down of hmox-1 gene expression by small interfering RNA or the blockade of HO-1 activity by zinc protoporphyrin IX abrogated the EHEC-dependent inhibition of STAT-1 activation and iNOS mRNA expression in activated human enterocytes. These results highlight a new strategy elaborated by EHEC to control the host innate immune response.