RESUMO
BACKGROUND: A role for pilus during attachment of Neisseria gonorrhoeae to epithelia of the female reproductive tract is currently assumed. However, Pilâ» gonococci have been observed during infection of the reproductive tract, which prompted us to examine the effect of pili on the dynamics of infection and the inflammatory responses of mucosal explants of the human fallopian tube. METHODS: Mucosal explants were infected in vitro with Opa negative Pilâ» and PilâºN. gonorrhoeae strains. RESULTS: Piliation enhanced gonococcal adherence to the epithelium within 3 h of infection (P < 0.05) but thereafter did not offer advantage to gonococci to colonize the epithelial cell surface (P > 0.05). No differences were found between the strains in numbers of gonococci inside epithelial cells. Pilâ» bacteria induced higher levels (P < 0.05) of IL-1ß, TNF-α, GM-CSF, MCP-1, and MIP-1ß than Pil⺠bacteria. There were no differences between both strains in LOS pattern, and Pil expression did not change after coincubation with mucosal strips. CONCLUSIONS: Results show that gonococcal invasion of the human fallopian tube can occur independently of pilus or Opa expression, and suggest that pilus, by inhibition of several key elements of the initial inflammatory response, facilitates sustained infection of this organ.
Assuntos
Epitélio/microbiologia , Tubas Uterinas/microbiologia , Fímbrias Bacterianas/genética , Neisseria gonorrhoeae/genética , Proteínas da Membrana Bacteriana Externa/genética , Citocinas/genética , Citocinas/metabolismo , Feminino , Regulação Bacteriana da Expressão Gênica , Humanos , Inflamação/metabolismo , Inflamação/microbiologia , Neisseria gonorrhoeae/crescimento & desenvolvimento , Neisseria gonorrhoeae/patogenicidade , Técnicas de Cultura de ÓrgãosRESUMO
In the present study, we investigated whether cellular damage, as demonstrated by lactate dehydrogenase (LDH) release in the human fallopian tube (FT) infected by Neisseria gonorrhoeae (Ngo), correlated with high levels of nitric oxide synthase (NOS) mRNA and enzyme activity. Infection with Ngo induced a significant increase (~35-fold) in mRNA transcripts of the inducible isoform of NOS. Paradoxically, a reduction in NOS enzyme activity was observed in infected cultures, suggesting that gonococcal infection possibly influences translation of iNOS mRNA to the enzyme. In addition, treatment with the NOS inhibitor TRIM did not prevent gonococcal-induced cellular damage. In contrast, the addition of the inhibitor L-NAME induced a 40% reduction in LDH release, which correlated with a ~50% reduction in gonococcal numbers. Moreover, treatment of normal FT explants with an exogenous NO donor, SNAP, did not induce significant cellular damage. Taken together, our data suggest that NO does not contribute to cellular damage during infection of the human FT with Neisseria gonorrhoeae.
Assuntos
Tubas Uterinas/microbiologia , L-Lactato Desidrogenase/metabolismo , Neisseria gonorrhoeae/enzimologia , Óxido Nítrico Sintase/metabolismo , Óxido Nítrico/metabolismo , RNA Mensageiro/metabolismo , Células Cultivadas , Tubas Uterinas/patologia , Feminino , Humanos , Fatores de TempoRESUMO
In the present study, we investigated whether cellular damage, as demonstrated by lactate dehydrogenase (LDH) release in the human fallopian tube (FT) infected by Neisseria gonorrhoeae (Ngo), correlated with high levels of nitric oxide synthase (NOS) mRNA and enzyme activity. Infection with Ngo induced a significant increase (~35-fold) in mRNA transcripts of the inducible isoform of NOS. Paradoxically, a reduction in NOS enzyme activity was observed in infected cultures, suggesting that gonococcal infection possibly influences translation of iNOS mRNA to the enzyme. In addition, treatment with the NOS inhibitor TRIM did not prevent gonococcal-induced cellular damage. In contrast, the addition of the inhibitor L-NAME induced a 40 percent reduction in LDH release, which correlated with a ~50 percent reduction in gonococcal numbers. Moreover, treatment of normal FT explants with an exogenous NO donor, SNAP, did not induce significant cellular damage. Taken together, our data suggest that NO does not contribute to cellular damage during infection of the human FT with Neisseria gonorrhoeae.
Assuntos
Feminino , Humanos , Tubas Uterinas/microbiologia , L-Lactato Desidrogenase/metabolismo , Neisseria gonorrhoeae/enzimologia , Óxido Nítrico Sintase/metabolismo , Óxido Nítrico/metabolismo , RNA Mensageiro/metabolismo , Células Cultivadas , Tubas Uterinas/patologia , Fatores de TempoRESUMO
BACKGROUND: Infection of the Fallopian tubes (FT) by Neisseria gonorrhoeae (Ngo) can lead to acute salpingitis, an inflammatory condition resulting in damage primarily to the ciliated cells, with loss of ciliary activity and sloughing of the cells from the epithelium. Recently, we have shown that Ngo infection induced apoptosis in FT epithelium cells by a TNF-alpha dependent mechanism that could contribute to the cell and tissue damage observed in gonococcal salpingitis. AIM: To investigate the apoptosis-related genes expressed during apoptosis induction in cultured FT epithelial cells infected in vitro by Ngo. MATERIALS AND METHODS: In the current study, we used cDNA macroarrays and real time PCR to identify and determine the expression levels of apoptosis related genes during the in vitro gonococci infection of FT epithelial cells. RESULTS: Significant apoptosis was induced following infection with Ngo. Macroarray analysis identified the expression of multiple genes of the TNF receptor family (TNFRSF1B, -4, -6, -10A, -10B and -10D) and the Bcl-2 family (BAK1, BAX, BLK, HRK and MCL-1) without differences between controls and infected cells. This lack of difference was confirmed by RT-PCR of BAX, Bcl-2, TNFRS1A (TNFR-I) and TNFRSF1B (TNFR-II). CONCLUSION: Several genes related to apoptosis are expressed in primary cultures of epithelial cells of the human Fallopian tube. Infection with Ngo induces apoptosis without changes in the pattern of gene expression of several apoptosis-related genes. RESULTS strongly suggest that Ngo regulates apoptosis in the FT by post-transcriptional mechanisms that need to be further addressed.
Assuntos
Apoptose/genética , Células Epiteliais/microbiologia , Tubas Uterinas/microbiologia , Neisseria gonorrhoeae/fisiologia , Salpingite/microbiologia , Células Cultivadas , Células Epiteliais/patologia , Tubas Uterinas/patologia , Feminino , Regulação da Expressão Gênica , Humanos , Análise de Sequência com Séries de Oligonucleotídeos , Proteínas Proto-Oncogênicas c-bcl-2/metabolismo , Receptores do Fator de Necrose Tumoral/metabolismo , Reação em Cadeia da Polimerase Via Transcriptase Reversa , Salpingite/patologiaRESUMO
Background: Infection of the Fallopian tubes (FT) by Neisseria gonorrhoeae (Ngo) can lead to acute salpingitis, an inflammatory condition resulting in damage primarily to the ciliated cells, with loss of ciliary activity and sloughing of the cells from the epithelium. Recently, we have shown that Ngo infection induced apoptosis in FT epithelium cells by a TNF-alpha dependent mechanism that could contribute to the cell and tissue damage observed in gonococcal salpingitis. Aim: To investigate the apoptosis-related genes expressed during apoptosis induction in cultured FT epithelial cells infected in vitro by Ngo. Materials and Methods: In the current study, we used cDNA macroarrays and real time PCR to identify and determine the expression levels of apoptosis related genes during the in vitro gonococci infection of FT epithelial cells. Results: Significant apoptosis was induced following infection with Ngo. Macroarray analysis identified the expression of multiple genes of the TNF receptor family (TNFRSF1B, -4, -6, -10A, -10B and -10D) and the Bcl-2 family (BAK1, BAX, BLK, HRK and MCL-1) without differences between controls and infected cells. This lack of difference was confirmed by RT-PCR of BAX, Bcl-2, TNFRS1A (TNFR-I) and TNFRSF1B (TNFR-II). Conclusion: Several genes related to apoptosis are expressed in primary cultures of epithelial cells of the human Fallopian tube. Infection with Ngo induces apoptosis without changes in the pattern of gene expression of several apoptosis-related genes. Results strongly suggest that Ngo regulates apoptosis in the FT by post-transcriptional mechanisms that need to be further addressed.
Assuntos
Feminino , Humanos , Apoptose/genética , Células Epiteliais/microbiologia , Tubas Uterinas/microbiologia , Neisseria gonorrhoeae/fisiologia , Salpingite/microbiologia , Células Cultivadas , Células Epiteliais/patologia , Tubas Uterinas/patologia , Regulação da Expressão Gênica , Análise de Sequência com Séries de Oligonucleotídeos , /metabolismo , Reação em Cadeia da Polimerase Via Transcriptase Reversa , Receptores do Fator de Necrose Tumoral/metabolismo , Salpingite/patologiaRESUMO
Following infection with Neisseria gonorrhoeae, bacteria may ascend into the Fallopian tubes (FT) and induce salpingitis, a major cause of infertility. In the FT, interactions between mucosal epithelial cells and gonococci are pivotal events in the pathogen's infection cycle and the inflammatory response. In the current study, primary FT epithelial cells were infected in vitro with different multiplicities of infection (MOI) of Pil+ Opa+ gonococci. Bacteria showed a dose-dependent association with cells and induced the secretion of tumor necrosis factor alpha (TNF-alpha). A significant finding was that gonococcal infection (MOI = 1) induced apoptosis in approximately 30% of cells, whereas increasing numbers of bacteria (MOI = 10 to 100) did not induce apoptosis. Apoptosis was observed in only 11% of cells with associated bacteria, whereas >84% of cells with no adherent bacteria were apoptotic. TNF-alpha was a key contributor to apoptosis, since (i) culture supernatants from cells infected with gonococci (MOI = 1) induced apoptosis in naïve cultures, suggesting that a soluble factor was responsible; (ii) gonococcal infection-induced apoptosis was inhibited with anti-TNF-alpha antibodies; and (iii) the addition of exogenous TNF-alpha induced apoptosis, which was inhibited by the presence of increasing numbers of bacteria (MOI = 10 to 100). These data suggest that TNF-alpha-mediated apoptosis of FT epithelial cells is likely a primary host defense mechanism to prevent pathogen colonization. However, epithelial cell-associated gonococci have evolved a mechanism to protect the cells from undergoing TNF-alpha-mediated apoptosis, and this modulation of the host innate response may contribute to establishment of infection. Understanding the antiapoptotic mechanisms used by Neisseria gonorrhoeae will inform the pathogenesis of salpingitis and could suggest new intervention strategies for prevention and treatment of the disease.
Assuntos
Apoptose , Tubas Uterinas/microbiologia , Neisseria gonorrhoeae/patogenicidade , Salpingite/etiologia , Fator de Necrose Tumoral alfa/fisiologia , Células Cultivadas , Células Epiteliais/microbiologia , Células Epiteliais/patologia , Tubas Uterinas/patologia , Feminino , Humanos , Imunidade Inata , Salpingite/imunologiaRESUMO
Infection of the Fallopian tubes (FT) by Neisseria gonorrhoeae can lead to acute salpingitis, an inflammatory condition, which is a major cause of infertility. Challenge of explants of human FT with gonococci induced mRNA expression and protein secretion for the proinflammatory cytokines interleukin (IL)-1alpha, IL-1beta, and tumor necrosis factor alpha (TNF-alpha) but not for granulocyte-macrophage colony-stimulating factor. In contrast, FT expression of IL-6 and of the cytokine receptors IL-6R, TNF receptor I (TNF-RI), and TNF-RII was constitutive and was not increased by gonococcal challenge. These studies suggest that several proinflammatory cytokines are likely to contribute to the cell and tissue damage observed in gonococcal salpingitis.