A critical role for hemolysin in Vibrio fluvialis-induced IL-β secretion mediated by the NLRP3 inflammasome in macrophages

Vibrio fluvialis causes human diarrhea, but the pathogenesis is not well-studied. We hypothesized that V fluvialis-secreted hemolysin (VFH) may induce IL-1 beta secretion through the activation of the NLRP3 inflammasome and contribute to the pathogenicity of V fluvialis. To examine this possibility, we constructed VFH mutant and complement strains and demonstrated that V fluvialls-induced IL-1 beta production and cytotoxicity in human monocytic THP-1 cells and mouse macrophages is attributed to VFH. To evaluate the role of VFH in vivo, we infected adult C57BL/6 mice intraperitoneally and suckling C57/B6 mice orally with various strains. The mice treated with 108 CFU wildtype V fluvialis or cell-free supernatant containing VFH induced significantly higher IL-1 production in peritoneal lavage fluid or in colon compared with those infected with the mutant strain, while no effect on TNF and IL-6 production was observed at day 5 or 24 h post-infection. VFH contributed to pathological changes and IL-1 release independent of colonization of V fluvialis in the colon. VFH has no effect on the synthesis of pro-IL-1 beta, but rather it triggers the processing of pro-IL-1 beta into IL-1 beta. Furthermore, using deficient mouse strains, we verified that V fluvialls-induced IL-1 beta is mediated through activation of Caspase-1 and the NLRP3 inflammasome ex vivo. Confocal microscopy suggests that VFH contributes to cathepsin B release. Furthermore, V fluvialis-induced IL-1 13 secretion requires potassium (K+) efflux and reactive oxygen species production. Our results provide new evidence for the role of VFH in the activation of the NLRP3 inflammasome and pathogenesis in response to V fluvialis infection. Summary Sentence: Vibrio fluvialis-secreted hemolysin induces IL-1 beta secretion through the activation of the NLRP3 inflammasome and contributes to the pathogenicity of V fluvialis.