Alain P. Gobert

Helicobacter pylori arginase inhibits nitric oxide production by eukaryotic cells: A strategy for bacterial survival

The antimicrobial effect of nitric oxide (NO) is an essential part of innate immunity. The vigorous host response to the human gastric pathogen Helicobacter pylori fails to eradicate the organism, despite up-regulation of inducible NO synthase (iNOS) in the gastric mucosa. Here we report that wild-type strains of H. pylori inhibit NO production by activated macrophages at physiologic concentrations of l-arginine, the common substrate for iNOS and arginase. Inactivation of the gene rocF, encoding constitutively expressed arginase in H. pylori, restored high-output NO production by macrophages. By using HPLC analysis, we show that l-arginine is effectively consumed in the culture medium by wild-type but not arginase-deficient H. pylori. The substantially higher levels of NO generated by macrophages cocultured with rocF-deficient H. pylori resulted in efficient killing of the bacteria, whereas wild-type H. pylori exhibited no loss of survival under these conditions. Killing of the arginase-deficient H. pylori was NO-dependent, because peritoneal macrophages from iNOS−/− mice failed to affect the survival of the rocF mutant. Thus, bacterial arginase allows H. pylori to evade the immune response by down-regulating eukaryotic NO production.

Helicobacter pylori Induces Macrophage Apoptosis by Activation of Arginase II

Helicobacter pylori infection induces innate immune responses in macrophages, contributing to mucosal inflammation and damage. Macrophage apoptosis is important in the pathogenesis of mucosal infections but has not been studied with H. pylori. NO derived from inducible NO synthase (iNOS) can activate macrophage apoptosis. Arginase competes with iNOS by converting l-arginine to l-ornithine. Since we reported that H. pylori induces iNOS in macrophages, we now determined whether this bacterium induces arginase and the effect of this activation on apoptosis. NF-κB-dependent induction of arginase II, but not arginase I, was observed in RAW 264.7 macrophages cocultured with H. pylori. The time course of apoptosis matched those of both arginase and iNOS activities. Surprisingly, apoptosis was blocked by the arginase inhibitors Nω-hydroxy-l-arginine or Nω-hydroxy-nor-l-arginine, but not by the iNOS inhibitor N-iminoethyl-l-lysine. These findings were confirmed in peritoneal macrophages from iNOS-deficient mice and were not dependent on bacterial-macrophage contact. Ornithine decarboxylase (ODC), which metabolizes l-ornithine to polyamines, was also induced in H. pylori-stimulated macrophages. Apoptosis was abolished by inhibition of ODC and was restored by the polyamines spermidine and spermine. We also demonstrate that arginase II expression is up-regulated in both murine and human H. pylori gastritis tissues, indicating the likely in vivo relevance of our findings. Therefore, we describe arginase- and ODC-dependent macrophage apoptosis, which implicates polyamines in the pathophysiology of H. pylori infection.

l-Arginine Availability Modulates Local Nitric Oxide Production and Parasite Killing in Experimental Trypanosomiasis

ABSTRACT Nitric oxide (NO) is an important effector molecule of the immune system in eliminating numerous pathogens. Peritoneal macrophages fromTrypanosoma brucei brucei-infected mice express type II NO synthase (NOS-II), produce NO, and kill parasites in the presence ofl-arginine in vitro. Nevertheless, parasites proliferate in the vicinity of these macrophages in vivo. The present study shows thatl-arginine availability modulates NO production. Trypanosomes use l-arginine for polyamine synthesis, required for DNA and trypanothione synthesis. Moreover, arginase activity is up-regulated in macrophages from infected mice from the first days of infection. Arginase competes with NOS-II for their common substrate, l-arginine. In vitro, arginase inhibitors decreased urea production, increased macrophage nitrite production, and restored trypanosome killing. In vivo, a dramatic decrease inl-arginine concentration was observed in plasma from infected mice. In situ restoration of NO production and trypanosome killing were observed when excess l-arginine, but notd-arginine or l-arginine plusNω-nitro-l-arginine (a NOS inhibitor), was injected into the peritoneum of infected mice. These data indicate the role of l-arginine depletion, induced by arginase and parasites, in modulating the l-arginine–NO pathway under pathophysiological conditions.

HELICOBACTER PYLORI HEAT SHOCK PROTEIN 60 MEDIATES INTERLEUKIN-6 PRODUCTION BY MACROPHAGES VIA A TOLL-LIKE RECEPTOR (TLR)-2-, TLR-4-, AND MYELOID DIFFERENTIATION FACTOR 88-INDEPENDENT MECHANISM

Helicobacter pylori has been reported to induce interleukin-6 (IL-6) production in monocytes/macrophages and in chronically inflamed gastric tissues. The mechanism by which H. pylori induces IL-6 production in macrophages, however, has not been investigated. To identify the H. pylori factor responsible for this activity, we fractionated soluble proteins from H. pylori strain 26695 by ion exchange and size exclusion chromatography and screened the fractions for IL-6-inducing activity on RAW 264.7 macrophages. A single protein was purified and identified by mass spectrometry as H. pylori heat shock protein 60 (HSP60). Consistent with the observed IL-6-inducing activity of H. pylori HSP60, soluble protein extracts of H. pylori 26695 and SS1 strains that were depleted of this protein by affinity chromatography had dramatically reduced IL-6-inducing activities. The immunopurified HSP60 stimulated IL-6 production in macrophages. When stimulated with H. pylori HSP60 or intact bacteria, peritoneal macrophages from mice deficient in Toll-like receptor (TLR)-2, TLR-4, TLR-2/TLR-4, and myeloid differentiation factor 88 produced the same amount of IL-6 than macrophages from wild-type mice, demonstrating the independence of H. pylori HSP60 responses from these signaling molecules. H. pylori HSP60-induced IL-6 mRNA expression, and NF-κB activation in RAW 264.7 cells was abrogated in the presence of MG-132, a proteasome inhibitor. In contrast, inhibitors of protein kinase A or C, mitogen-activated protein kinase kinase, and phosphoinositide 3-kinase had no effect on IL-6 mRNA levels. This study demonstrates the induction of innate immune responses by H. pylori HSP60, thereby implicating this highly conserved protein in the pathophysiology of chronic gastritis.

Arginases in parasitic diseases

Abstract Parasites have elaborated a variety of strategies for invading hosts and escaping immune responses. This article proposes that a common mechanism whereby different parasites escape nitric oxide (NO) toxicity is the activation of arginase. This leads to a depletion of l-arginine (substrate of NO synthase, resulting in lower levels of cytotoxic NO) and increased production of polyamines (necessary for parasite growth and differentiation).

Cutting Edge: Urease Release by Helicobacter pylori Stimulates Macrophage Inducible Nitric Oxide Synthase

Inducible NO synthase (iNOS) expression and production of NO are both up-regulated with Helicobacter pylori infection in vivo and in vitro. We determined whether major pathogenicity proteins released by H. pylori activate iNOS by coculturing macrophages with wild-type or mutant strains deficient in VacA, CagA, picB product, or urease (ureA−). When filters were used to separate H. pylori from macrophages, there was a selective and significant decrease in stimulated iNOS mRNA, protein, and NO2− production with the ureA− strain compared with wild-type and other mutants. Similarly, macrophage NO2− generation was increased by H. pylori protein water extracts of all strains except ureA−. Recombinant urease stimulated significant increases in macrophage iNOS expression and NO2− production. Taken together, these findings indicate a new role for the essential H. pylori survival factor, urease, implicating it in NO-dependent mucosal damage and carcinogenesis.

Protective Role of Arginase in a Mouse Model of Colitis

Arginase is the endogenous inhibitor of inducible NO synthase (iNOS), because both enzymes use the same substrate, l-arginase (Arg). Importantly, arginase synthesizes ornithine, which is metabolized by the enzyme ornithine decarboxylase (ODC) to produce polyamines. We investigated the role of these enzymes in the Citrobacter rodentium model of colitis. Arginase I, iNOS, and ODC were induced in the colon during the infection, while arginase II was not up-regulated. l-Arg supplementation of wild-type mice or iNOS deletion significantly improved colitis, and l-Arg treatment of iNOS−/− mice led to an additive improvement. There was a significant induction of IFN-γ, IL-1, and TNF-α mRNA expression in colitis tissues that was markedly attenuated with l-Arg treatment or iNOS deletion. Treatment with the arginase inhibitor S-(2-boronoethyl)-l-cysteine worsened colitis in both wild-type and iNOS−/− mice. Polyamine levels were increased in colitis tissues, and were further increased by l-Arg. In addition, in vivo inhibition of ODC with α-difluoromethylornithine also exacerbated the colitis. Taken together, these data indicate that arginase is protective in C. rodentium colitis by enhancing the generation of polyamines in addition to competitive inhibition of iNOS. Modulation of the balance of iNOS and arginase, and of the arginase-ODC metabolic pathway may represent a new strategy for regulating intestinal inflammation.

Human Microbiota-Secreted Factors Inhibit Shiga Toxin Synthesis by Enterohemorrhagic Escherichia coli O157:H7

ABSTRACT 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 stx2 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 stx2 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.

Shiga toxin produced by enterohemorrhagic Escherichia coli inhibits PI3K/NF-κB signaling pathway in globotriaosylceramide-3-negative human intestinal epithelial cells

Shiga toxin (Stx) produced by enterohemorrhagic Escherichia coli (EHEC) binds to endothelial cells expressing globotriaosylceramide-3 (Gb-3) and induces cell death by inhibiting translation. Nonetheless, the effects of Stx on human enterocytes, which lacks receptor Gb-3, remain less known. In this study, we questioned whether EHEC-derived Stx may modulate cellular signalization in the Gb-3-negative human epithelial cell line T84. Stx produced by EHEC was fixed and internalized by the cells. A weak activation of NF-κB was observed in T84 cells after EHEC infection. Cells infected with an isogenic mutant lacking stx1 and stx2, the genes encoding Stx, displayed an increased NF-κB DNA-binding activity. Consequently, the NF-κB-dependent CCL20 and IL-8 gene transcription and chemokine production were enhanced in T84 cells infected with the Stx mutant in comparison to the wild-type strain. Investigating the mechanism by which Stx modulates NF-κB activation, we showed that the PI3K/Akt signaling pathway was not induced by EHEC but was enhanced by the strain lacking Stx. Pharmacological inhibition of the PI3K/Akt signalization in EHEC ΔStx-infected T84 cells yielded to a complete decrease of NF-κB activation and CCL20 and IL-8 mRNA expression. This demonstrates that the induction of the PI3K/Akt/NF-κB pathway is potentially induced by EHEC, but is inhibited by Stx in Gb-3-negative epithelial cells. Thus, Stx is an unrecognized modulator of the innate immune response of human enterocytes.

Cutting edge: cyclooxygenase-2 activation suppresses Th1 polarization in response to Helicobacter pylori.

Helicobacter pylori infection causes a Th1-driven mucosal immune response. Cyclooxygenase (COX)-2 is up-regulated in lamina propria mononuclear cells in H. pylori gastritis. Because COX-2 can modulate Th1/Th2 balance, we determined whether H. pylori activates COX-2 in human PBMCs, and the effect on cytokine and proliferative responses. There was significant up-regulation of COX-2 mRNA and PGE2 release in response to H. pylori preparations. Addition of COX-2 inhibitors or an anti-PGE2 Ab resulted in a marked increase in H. pylori-stimulated IL-12 and IFN-γ production, and a decrease in IL-10 levels. Addition of PGE2 or cAMP, the second messenger activated by PGE2, had the opposite effect. Similarly, stimulated cell proliferation was increased by COX-2 inhibitors or anti-PGE2 Ab, and was decreased by PGE2. Our findings indicate that COX-2 has an immunosuppressive role in H. pylori gastritis, which may protect the mucosa from severe injury, but may also contribute to the persistence of the infection.