Although there is a growing body of evidence regarding the virulence factors of EHEC O157:H7, such as Stxs and flagellin in epithelial cells, the role of specific Ehx encoding on plasmid of EHEC O157:H7 in pathogenesis has not been fully elucidated. It is likely that the EHEC-Ehx is expressed during human infection and subsequent disease, as patients suffering from O157-associated HUS produce specific EHEC-Ehx antibodies in almost all cases 
The EHEC-Ehx is a highly active repeats-in-toxin with pore-forming capacity similar but not identical to that of chromosomal encoded E. coli
α-hemolysin. The presence of α-hemolysin in enteroaggregative and cytodetaching Escherichia coli
strains appears to play a critical role in both oncosis in human monocyte-derived macrophages and apoptosis in the murine macrophage cell line (J774 cells) 
. The hemolysin A of E. coli
was found to increase the permeability of human macrophages by forming ionic pores 
. Bauer and Welch found that EHEC-Ehx lysed bovine but not human lymphoma cells. They hypothesized that the target cell specificity of EHEC-Ehx might be narrow 
. Kartch’s group has reported that the EHEC-Ehx is cytotoxic to human brain microvascular endothelial cells and that this toxicity may contribute to the virulence of the stx
-negative E. coli
O26 strains 
. Our data provide clear evidence that EHEC-Ehx encoded on the plasmid of EDL933 contributed to the cytotoxicity of EHEC in THP-1 cells. Macrophages are the main producers of proinflammatory cytokines in response to bacterial infection and the cytotoxicity of the macrophages can affect the host immune response to bacterial invasion and affect the pathogenesis of EHEC O157:H7 infection.
Previous studies have shown that the inflammatory response is involved in the pathogenesis of EHEC O157:H7 infection 
. HUS patients show an increase in a variety of circulating proinflammatory cytokines, such as IL-1β, TNF-α, and IL-8, in response to EHEC O157:H7 infection 
. However, which components of EHEC O157:H7 contribute to the elevated level of specific pro-inflammatory cytokines through macrophage activity has not been well demonstrated. In this study, we demonstrated that the EHEC-Ehx induced a higher level of mature IL-1β in THP-1 cells. Other cytokines (IL-6, IL-8, RANETS/CCL5, MCP-1, TNF-α, and IFN-γ) were also examined and none of them were induced by Ehx.
IL-1β is an important proinflammatory mediator. It exerts a variety of biological effects. During EHEC O157:H7 infection, IL-1β is a potent inducer of fever and inflammatory response. It can disrupt the intestinal barrier, permitting transport of Stxs into the circulatory system 
. IL-1β was also found to be involved in HUS through increasing expression of Gb3, the receptor of Stx on endothelial cells allowing increased binding of Stx 
. In this study, we observed that EHEC-Ehx could contribute to the release of mature IL-1β by THP-1 cells.
To determine the mechanism underlying the EHEC O157:H7-Ehx-induced release of IL-1β, we investigated how Ehx might play a role in each step of the release of IL-1β. The mechanism underlying the release of IL-1β has three major steps: 1) Synthesis the biologically inactive pro-IL-1β. 2) Cleavage of pro-IL-1β by caspase-1 processing into mature biologically active IL-1β. 3) Secretion of mature IL-1β into extracellular milieu 
. First, we found that Ehx had no effect on intracellular gene expression and production of biologically inactive pro-IL-1β in THP-1 cells by RT-PCR and immunoblotting. These data imply that EhxA may affect the subsequent steps in the release of IL-1β release. Second, we demonstrated that the NLRP3/ASC/caspase-1 inflammasome is required for EHEC O157:H7-induced IL-1β production using RNA interference experiments. The cysteine protease caspase-1 is responsible for the proteolytic processing and secretion of IL-1β. 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 
. In this study, although our current data demonstrated that EHEC O157:H7-induced Il-1β 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-1β 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-1β. Third, different exocytosis pathways have been observed in monocytes, macrophages, and dendritic cells. These pathways export the cytokine IL-1β, one of which is the type of IL-1β released upon cell lysis 
. In this study, we found a positive correlation between IL-1β production and cytotoxicity induced by EHEC-Ehx. Even the cytotoxicity of Ehx has been found to contribute to the release of IL-1β through cell lysis, which cannot be the main source of extracellular IL-1β because most of the IL-1β in the supernatant was biologically active mature IL-1β, as shown by immunoblot analysis. Further experiments are needed to determine the mechanism by which cytotoxicity of Ehx affects the secretion of mature IL-1β into the extracellular space and how cytotoxic Ehx affects the pathogenesis of EHEC infection.
In 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-1β. The NLRP3 inflammasome was found to mediate EHEC O157:H7-activated IL-1β production. Ehx may activate pro-caspase-1 through activation of NLRP3, like other pore-form bacteria toxins. However, the possibility that other types of inflammasome signaling may be activated by Ehx cannot yet be ruled out. This may also have stimulated the release of IL-1β. Cytotoxicity to THP-1 cells may also contribute to the release of IL-1β using some as yet unknown mechanism. Further study is needed to determine the possible roles of IL-1β in the pathogenesis of this potentially fatal foodborne infection.