Proinflammatory cytokine and nitric oxide production by human macrophages stimulated with Trichomonas vaginalis.

Trichomonas vaginalis commonly causes vaginitis and perhaps cervicitis in women and urethritis in men and women. Macrophages are important immune cells in response to T. vaginalis infection. In this study, we investigated whether human macrophages could be involved in inflammation induced by T. vaginalis. Human monocyte-derived macrophages (HMDM) were co-cultured with T. vaginalis. Live, opsonized-live trichomonads, and T. vaginalis lysates increased proinflammatory cytokines, such as TNF-alpha, IL-1beta, and IL-6 by HMDM. The involvement of nuclear factor (NF)-kappaB signaling pathway in cytokine production induced by T. vaginalis was confirmed by phosphorylation and nuclear translocation of p65 NF-kappaB. In addition, stimulation with live T. vaginalis induced marked augmentation of nitric oxide (NO) production and expression of inducible NO synthase (iNOS) levels in HMDM. However, trichomonad-induced NF-kappaB activation and TNF-alpha production in macrophages were significantly inhibited by inhibition of iNOS levels with L-NMMA (NO synthase inhibitor). Moreover, pretreatment with NF-kappaB inhibitors (PDTC or Bay11-7082) caused human macrophages to produce less TNF-alpha. These results suggest that T. vaginalis stimulates human macrophages to produce proinflammatory cytokines, such as IL-1, IL-6, and TNF-alpha, and NO. In particular, we showed that T. vaginalis induced TNF-alpha production in macrophages through NO-dependent activation of NF-kappaB, which might be closely involved in inflammation caused by T. vaginalis.

Vibrio vulnificus-induced cell death of human mononuclear cells requires ROS-dependent activation of p38 and ERK 1/2 MAPKs.

Vibrio vulnificus is a Gram-negative bacterium that multiplies rapidly in host tissue and causes extensive tissue damage. Human peripheral blood mononuclear cells (PBMC) were shown to be readily killed by exposure to live V. vulnificus. V. vulnificus induced production of intracellular reactive oxygen species (ROS) and nitric oxide (NO) in PBMC. Pretreatment of PBMC with diphenyleneiodonium chloride (DPI) abolished ROS generation upon exposure to V. vulnificus and decreased the bacterial ability to cause cell death. In contrast, pretreatment of these cells with inhibitors of inducible nitric oxide synthase (iNOS) blocked V. vunificus-induced NO production, but did not significantly alter cell death by V. vulnificus. V. vulnificus also triggered phosphorylation of mitogen-activated protein kinases (MAPKs), including p38 and ERK1/2 in PBMC. Inactivation of these MAPKs by selective inhibitors caused a reduction both in ROS generation and cell death induced by V. vulnificus. It was further shown that an inhibitor of ROS generation (DPI) blocked V. vulnificus-induced phosphorylation of p38 and ERK1/2 MAPK. This study demonstrates that V. vulnificus induces death of PBMC via ROS-dependent activation of p38 MAPK and ERK1/2 MAPK.

Vibrio vulnificus-induced death of Jurkat T-cells requires activation of p38 mitogen-activated protein kinase by NADPH oxidase-derived reactive oxygen species.

Vibrio vulnificus, a pathogenic bacterium causing primary septicemia, exhibited cytotoxicity towards Jurkat cells of T-lymphocytes through intracellular reactive oxygen species (ROS) production. Pretreatment of Jurkat T-cells with diphenyleneiodonium chloride (DPI) abolished V. vulnificus-induced ROS generation and bacterial ability to cause cell death. Jurkat T-cells expressing dominant-negative protein of Rac subunit of NADPH oxidase (NOX) did not show increased ROS production and cell death by V. vulnificus. Vibrio vulnificus also triggered phosphorylation of mitogen-activated protein kinases (MAPKs) including p38 and ERK1/2 in Jurkat T-cells. Experiments using inhibitors or small interfering RNAs for each MAPK showed that both MAPKs are involved in V. vulnificus-induced cell death. DPI only blocked the phosphorylation of p38 MAPK in Jurkat T-cells exposed by V. vulnificus. This study demonstrates that V. vulnificus induces death of Jurkat T-cells via ROS-dependent activation of p38 MAPK, and that NOX plays a major role in ROS generation in V. vulnificus-exposed cells.

Vibrio vulnificus IlpA-induced cytokine production is mediated by Toll-like receptor 2.

Vibrio vulnificus is a pathogenic bacterium causing primary septicemia, which follows a classical septic shock pathway, including an overwhelming inflammatory cytokine response. In this study, we identified a putative lipoprotein of V. vulnificus, encoded by the ilpA gene, as one of the surface proteins that specifically reacted with the antibodies raised against outer membrane proteins of V. vulnificus. Using a mutant V. vulnificus in which its ilpA gene was knocked out, we found that IlpA is important in the production of interferon-gamma in human peripheral blood mononuclear cells. Production of tumor necrosis factor-alpha and interleukin-6 is also induced by the recombinant IlpA (rIlpA) in human monocytes. Lipidation of the rIlpA was observed by in vivo labeling in Escherichia coli. Experiments using the mutant IlpA, which is unable to be modified by lipidation, indicate that the lipid moiety of this protein has an essential property for cytokine production in human cells. Pretreatment of monocytes with antibodies against Toll-like receptor 2 (TLR2) inhibited production of both tumor necrosis factor-alpha and interleukin-6. The role of TLR2 in IlpA-induced cytokine production was confirmed by an in vitro assay, in which only the TLR2-expressing cells showed a dramatic induction of nuclear factor-kappaB activity by rIlpA. In addition, rIlpA treatment resulted in induction of TLR2 transcription in human cells. In comparison with the wild type V. vulnificus, the ilpA mutant showed a reduced mortality in mice. These results demonstrate that IlpA of V. vulnificus functions as an immunostimulant to human cells via TLR2.

Identification of OmpU of Vibrio vulnificus as a fibronectin-binding protein and its role in bacterial pathogenesis.

Vibrio vulnificus is a pathogenic bacterium that causes gastroenteritis and primary septicemia. To identify factors involved in microbial adherence to the host cells, we investigated bacterial proteins capable of binding to fibronectin, one of the main components comprised of the extracellular matrix of mammalian cells. A protein of approximately 35 kDa was purified from the extracts of V. vulnificus by its property to bind to immobilized fibronectin. This protein was identified as OmpU, one of the major outer membrane proteins of V. vulnificus. In binding assays using immobilized fibronectin, the number of ompU mutant cells bound to fibronectin was only 4% of that of wild-type cells bound to fibronectin. In addition, the exogenous addition of antibodies against OmpU resulted in a decreased ability of wild-type V. vulnificus to adhere to fibronectin. The ompU mutant was also defective in its adherence to RGD tripeptide (5% of the adherence of the wild type to RGD), cytoadherence to HEp-2 cells (7% of the adherence of the wild type to HEp-2), cytotoxicity to cell cultures (39% of the cytotoxicity of the wild type), and mortality in mice (10-fold increase in the 50% lethal dose). The ompU mutant complemented with the intact ompU gene restored its abilities for adherence to fibronectin, RGD tripeptide, and HEp-2 cells; cytotoxicity to HEp-2 cells; and mouse lethality. This study indicates that OmpU is an important virulence factor involved in the adherence of V. vulnificus to the host cells.