Vibrio vulnificus cytolysin induces inflammatory responses in RAW264.7 macrophages through calcium signaling and causes inflammation in vivo

Abstract

Vibrio vulnificus is a food-borne marine pathogen that causes both life-threatening primary septicemia and necrotizing wound infections which accompany severe inflammation. Cytolysin is a very powerful virulence factor of V. vulnificus and is one of the likely candidates in the pathogenesis of V. vulnificus infections. However, the pathogenetic roles of cytolysin in V. vulnificus-induced inflammation are not well understood. In this study, we used the recombinant protein Vibrio vulnificus cytolysin (VVC) to demonstrate that VVC can induce inflammatory responses in RAW264.7 macrophages. Low dose (<5 μg/ml) VVC had no impact on cell viability and induced pro-inflammatory cytokines production in RAW264.7 macrophages such as IL-6 and TNF-α. Moreover, VVC induced p65, p38, ERK1/2, and AKT phosphorylation in RAW264.7 macrophages. We further demonstrated that BAPTA-AM, a specific intracellular calcium chelator, inhibited VVC-induced inflammatory responses including pro-inflammatory cytokines production and inflammatory signaling activation in RAW264.7 macrophages. In addition, VVC primed rather than actived NLRP3 inflammasome in RAW264.7 macrophages. To determine whether VVC have a direct inflammatory effect on the host, we examined the effects of VVC injected into the skin of mice. VVC stimulated a significant induction of mRNAs for the pro-inflammatory cytokine IL-6 and inflammatory chemokines such as MCP-1 and IP-10. Histology data also showed that VVC caused inflammatory responses in the skin of mice. Collectively, our findings indicated that VVC induced inflammatory responses in RAW264.7 macrophages and in vivo and suggested the possibility of targeting VVC as a strategy for the clinical management of V. vulnificus-induced inflammatory responses.