RESUMO
Helicobacter pylori infection of the stomach elicits a vigorous but ineffective host immune and inflammatory response, resulting in persistence of the bacterium for the life of the host. We have reported that in macrophages, H. pylori up-regulates inducible NO synthase (iNOS) and antimicrobial NO production, but in parallel there is induction of arginase II, generating ornithine, and of ornithine decarboxylase (ODC), generating polyamines. Spermine, in particular, has been shown to restrain immune response in activated macrophages by inhibiting proinflammatory gene expression. We hypothesized that spermine could prevent the antimicrobial effects of NO by inhibiting iNOS in macrophages activated by H. pylori. Spermine did not affect the up-regulation of iNOS mRNA levels but in a concentration-dependent manner significantly attenuated iNOS protein levels and NO production. Reduction in iNOS protein was due to inhibition of iNOS translation and not due to iNOS degradation. ODC knockdown with small interfering (si) RNA resulted in increased H. pylori-stimulated iNOS protein expression and NO production without altering iNOS mRNA levels. When macrophages were cocultured with H. pylori, killing of bacteria was enhanced by transfection of ODC siRNA and prevented by addition of spermine. These results identify a mechanism of immune dysregulation induced by H. pylori in which stimulated spermine synthesis by the arginase-ODC pathway inhibits iNOS translation and NO production, leading to persistence of the bacterium and risk for peptic ulcer disease and gastric cancer.
Assuntos
Helicobacter pylori/imunologia , Óxido Nítrico Sintase/metabolismo , Biossíntese de Proteínas , Espermina/farmacologia , Animais , Apoptose , Linhagem Celular , Técnicas de Cocultura , Relação Dose-Resposta a Droga , Helicobacter pylori/metabolismo , Sistema Imunitário/metabolismo , Immunoblotting , Imunoprecipitação , Macrófagos/imunologia , Macrófagos/metabolismo , Macrófagos/microbiologia , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Monócitos/metabolismo , Óxido Nítrico/metabolismo , Óxido Nítrico Sintase Tipo II , RNA Interferente Pequeno/metabolismo , Reação em Cadeia da Polimerase Via Transcriptase Reversa , Risco , Espermina/metabolismo , Neoplasias Gástricas/microbiologia , Transfecção , Regulação para CimaRESUMO
Arginase is the endogenous inhibitor of inducible NO synthase (iNOS), because both enzymes use the same substrate, l-arginine (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-gamma, IL-1, and TNF-alpha 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 alpha-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.
Assuntos
Arginase/fisiologia , Colite/enzimologia , Infecções por Enterobacteriaceae/enzimologia , Óxido Nítrico Sintase/biossíntese , Ornitina Descarboxilase/fisiologia , Animais , Arginase/antagonistas & inibidores , Arginase/biossíntese , Arginase/genética , Arginina/metabolismo , Arginina/uso terapêutico , Ácidos Borônicos/farmacologia , Ácidos Borônicos/toxicidade , Citrobacter rodentium , Colite/tratamento farmacológico , Colite/microbiologia , Colite/patologia , Eflornitina/farmacologia , Eflornitina/toxicidade , Infecções por Enterobacteriaceae/microbiologia , Infecções por Enterobacteriaceae/patologia , Indução Enzimática , Inibidores Enzimáticos/farmacologia , Inibidores Enzimáticos/toxicidade , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Knockout , Óxido Nítrico Sintase/antagonistas & inibidores , Óxido Nítrico Sintase/genética , Óxido Nítrico Sintase Tipo II , Ornitina/metabolismo , Ornitina Descarboxilase/biossíntese , Ornitina Descarboxilase/genética , Poliaminas/metabolismoRESUMO
This review covers progress in identifying Helicobacter pylori-derived factors that are involved in survival and virulence of the organism and in elucidating host response pathways that can limit the infection but are also susceptible to dysregulation. Recent work has identified genes of the cytotoxin-associated gene (cag) pathogenicity island (PAI) involved in regulating signaling, interleukin-8 secretion, and phenotypic events in epithelial cells. New roles in pathogenesis have been recognized for vacuolating toxin A (VacA) and urease, H. pylori membrane and secreted factors, and host epithelial surface molecules. Molecular pathways involved in H. pylori-induced apoptosis in epithelial cells, T cells, and macrophages are being dissected. Activation of toll-like receptors and bacterial factors involved in nitric oxide (NO) and reactive oxygen species induction were also described. The ability of H. pylori to limit NO production by several mechanisms may be an important part of its ability to evade the host immune response.
RESUMO
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 NO(2)(-) production with the ureA(-) strain compared with wild-type and other mutants. Similarly, macrophage NO(2)(-) generation was increased by H. pylori protein water extracts of all strains except ureA(-). Recombinant urease stimulated significant increases in macrophage iNOS expression and NO(2)(-) 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.