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 Pleuromutilin 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 HIV-RT inhibitor 1 site 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.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.
