Ed in HUS through increasing expression of Gb3, the receptor of

July 20, 2017

Ed in HUS through increasing expression of Gb3, the receptor of Stx on endothelial cells allowing increased binding of Stx [3,34]. In this study, we observed that EHEC-Ehx could contribute to the release of mature IL-1b by THP-1 cells. To determine the mechanism underlying the EHEC O157:H7Ehx-induced release of IL-1b, we investigated how Ehx might play a role in each step of the release of IL-1b. The mechanism underlying the release of IL-1b has three major steps: 1) Synthesis the biologically inactive pro-IL-1b. 2) Cleavage of pro-IL-1b by caspase-1 processing into mature biologically active IL-1b. 3) 15900046 Secretion of mature IL-1b into extracellular milieu [35]. First, we found that Ehx had no effect on intracellular gene expression and production of biologically inactive pro-IL-1b 1326631 in THP-1 cells by RT-PCR and immunoblotting. These data imply that EhxA may affect the subsequent steps in the release of IL-1b release. 69056-38-8 Second, we demonstrated that the NLRP3/ASC/caspase-1 inflammasome is required for EHEC O157:H7-induced IL-1b production using RNA interference experiments. The cysteine protease caspase-1 is responsible for the proteolytic processing and secretion of IL-1b. The inflammasome is a multi-protein complex critical to the activation of caspase-1 and induction of inflammatory responses. The inflammasome complex includes at least one NLR and an adaptor protein called ASC, which links the NLR to procaspase-1. The NLRP3 inflammasome has been reported to be activated by bacterial pore-forming toxins [36?0]. In this study, although our current data demonstrated that EHEC O157:H7-induced Il-1b was only partially dependent on caspase-1/ASC/NLRP3 inflammasome, the evidence was not sufficient to support the conclusion that EHEC O157:H7 could induce the release of IL-1b through any caspase-1-dependent or -independent pathway. This is because neither caspase-1 nor ASC nor NLRP3 was completely silenced in these assays. Further experiments using gene knock-out mice are necessary to determine the role of these inflammasomes in EHEC-induced IL-1b. Third, different exocytosis pathways have been observed in monocytes, macrophages, and dendritic cells. These pathways export the cytokine IL-1b, one of which is the type of IL-1b released upon cell lysis [41]. In this study, we found a positive correlation between IL-1b production and cytotoxicity induced by EHEC-Ehx. Even the cytotoxicity of Ehx has been found to contribute to the release of IL-1b through cell lysis, which cannot be the main source of extracellular IL-1b because most of the IL-1b in the supernatant was biologically active mature IL-1b, as shown by immunoblot analysis. Further experiments are needed to determine the mechanism by which cytotoxicity of Ehx affects the secretion of mature IL-1b into the extracellular space and how cytotoxic Ehx affects the pathogenesis of EHEC infection.Enterohemolysin Induced Release of IL-1bFigure 7. Correlation between the release of LDH and concentration of IL-1b in THP-1 cells infected with EHEC O157:H7. A significant positive correlation was observed (P,0.01). doi:10.1371/JI 101 journal.pone.0050288.gIn this study, we found EHEC O157:H7-Ehx to contribute to cytotoxicity in THP-1 cells. It was also found responsible for higher levels of mature IL-1b. The NLRP3 inflammasome was found to mediate EHEC O157:H7-activated IL-1b production. Ehx may activate pro-caspase-1 through activation of NLRP3, like other pore-form bacteria toxins. However, the possibility that.Ed in HUS through increasing expression of Gb3, the receptor of Stx on endothelial cells allowing increased binding of Stx [3,34]. In this study, we observed that EHEC-Ehx could contribute to the release of mature IL-1b by THP-1 cells. To determine the mechanism underlying the EHEC O157:H7Ehx-induced release of IL-1b, we investigated how Ehx might play a role in each step of the release of IL-1b. The mechanism underlying the release of IL-1b has three major steps: 1) Synthesis the biologically inactive pro-IL-1b. 2) Cleavage of pro-IL-1b by caspase-1 processing into mature biologically active IL-1b. 3) 15900046 Secretion of mature IL-1b into extracellular milieu [35]. First, we found that Ehx had no effect on intracellular gene expression and production of biologically inactive pro-IL-1b 1326631 in THP-1 cells by RT-PCR and immunoblotting. These data imply that EhxA may affect the subsequent steps in the release of IL-1b release. Second, we demonstrated that the NLRP3/ASC/caspase-1 inflammasome is required for EHEC O157:H7-induced IL-1b production using RNA interference experiments. The cysteine protease caspase-1 is responsible for the proteolytic processing and secretion of IL-1b. The inflammasome is a multi-protein complex critical to the activation of caspase-1 and induction of inflammatory responses. The inflammasome complex includes at least one NLR and an adaptor protein called ASC, which links the NLR to procaspase-1. The NLRP3 inflammasome has been reported to be activated by bacterial pore-forming toxins [36?0]. In this study, although our current data demonstrated that EHEC O157:H7-induced Il-1b was only partially dependent on caspase-1/ASC/NLRP3 inflammasome, the evidence was not sufficient to support the conclusion that EHEC O157:H7 could induce the release of IL-1b through any caspase-1-dependent or -independent pathway. This is because neither caspase-1 nor ASC nor NLRP3 was completely silenced in these assays. Further experiments using gene knock-out mice are necessary to determine the role of these inflammasomes in EHEC-induced IL-1b. Third, different exocytosis pathways have been observed in monocytes, macrophages, and dendritic cells. These pathways export the cytokine IL-1b, one of which is the type of IL-1b released upon cell lysis [41]. In this study, we found a positive correlation between IL-1b production and cytotoxicity induced by EHEC-Ehx. Even the cytotoxicity of Ehx has been found to contribute to the release of IL-1b through cell lysis, which cannot be the main source of extracellular IL-1b because most of the IL-1b in the supernatant was biologically active mature IL-1b, as shown by immunoblot analysis. Further experiments are needed to determine the mechanism by which cytotoxicity of Ehx affects the secretion of mature IL-1b into the extracellular space and how cytotoxic Ehx affects the pathogenesis of EHEC infection.Enterohemolysin Induced Release of IL-1bFigure 7. Correlation between the release of LDH and concentration of IL-1b in THP-1 cells infected with EHEC O157:H7. A significant positive correlation was observed (P,0.01). doi:10.1371/journal.pone.0050288.gIn this study, we found EHEC O157:H7-Ehx to contribute to cytotoxicity in THP-1 cells. It was also found responsible for higher levels of mature IL-1b. The NLRP3 inflammasome was found to mediate EHEC O157:H7-activated IL-1b production. Ehx may activate pro-caspase-1 through activation of NLRP3, like other pore-form bacteria toxins. However, the possibility that.