It has been reported that several clinical strains of V. vulnificus induce apoptosis in human and murine macrophage cell lines [10, 31]. In this study, we showed that VVH could induce the death of murine primary macrophages. We further showed that murine macrophages from different organs display differential sensitivities to VVH-induced cell death. Liver Kupffer cells, splenic macrophages, and BMMφs are more sensitive to the cytotoxicity of VVH, while alveolar macrophages, lung interstitial macrophages, and lung neutrophils are resistant to VVH-induced cell death. The mechanisms that dictate the sensitivities of these cells towards VVH are unclear. One possibility is that VVH may partially inhibit innate immune responses in the spleen and liver by inducing macrophage death in these organs at an early stage of V. vulnificus infection.
To its hemolytic and cytolytic activities, the effects of VVH on immune cells have not been well defined. Previous studies have shown that pathogens' hemolysin could affect the host cells by triggering various cellular events, such as cell death or ROS production. Streptolysin O (SLO) triggers cell death through apoptosis in macrophages and neutrophils [32], and SLO also activates p38 via ASK1 and ROS [33]. Like SLO, Listeriolysin O(LLO)also triggers apoptosis in dendritic cells and T lymphocytes [34]. Moreover, nitric oxide (NO) production depends on high doses of pneumolysin (PLY) treatment and phagocytosis. However, the low dose of PLY mainly induces rapid ROS production [35]. Other hemolysins, such as hemolysin II (HlyII) of Bacillus cereus, have also been reported could induce apoptosis in host monocytes and macrophages in vivo [36]. During phagocytosis, the phagocytes internalized the microbes, such as L. interrogans that produce rSph2 hemolysin, which could directly target the mitochondria, and trigger cell apoptosis by induction of ROS and mitochondrial membrane damage [37]. This study found that the low dose of VVH could induce significant ROS production even without triggering apoptosis. To date, only the high dose of VVH has been reported to induce ROS production in epithelial cells and endothelial cells upon apoptosis [14, 15, 24, 38, 39]. The high dose of VVH induced cell death and ROS production through phosphorylation of a distinct ERK1/2 kinase [15, 25], and ERK1/2 did not respond to treatment with the low dose of VVH in murine BMMφs. We found that the low dose of VVH treatment elevated phosphorylation of p38, Akt, NFκB, and IKKα/β in murine BMMφs, and that low dose VVH triggered both p38-MAPKs- and NFκB-dependent ROS generation. Thus, the mechanisms of ROS production are likely different between low dose of VVH and high dose of VVH treatment.
TNF-α and IL-1 are two important mediators leading to sepsis. Previous studies have reported that VVH can induce the release of IL-1β, but not TNF-α in macrophages [40]. In this study, we found that a low dose of VVH can directly induce TNF-α expression. Our result is in line with a previous observation that VVH treatment increases TNF-α and IL-10 levels in murine serum [16]. For other pathogens, previous reports show that Leptospiral hemolysin induces TNF-α, IL-1β, and IL-6 levels in murine macrophages via toll-like receptor 2 (TLR2)- or TLR4-mediated JNK and NFκB pathways [41]. Sub-cytocidal concentrations of α-hemolysin from E. coli could lead to increased IL-1β production, but not that of TNF-α in monocytes [42]. Another hemolysin study reported that it was associated with triggering inflammation. SLO inhibited TNF-α and IL-1β release from infected macrophages to blunt macrophages' immune response [32]. LLO causes IL-1β production by activating NLRP3 inflammasome in macrophages [43] and inducing cytokine gene expression, including IL-1, TNF-α, IFN-γ, and IL-12 [44]. However, the expression of IL-1β, IL-6, and IL-8 was not affected by PLY. Only the TNF-α level was enhanced by PLY in a phagocytosis-independent manner [35]. Vibrio parahaemolyticus plays a major role in triggering NLRP3 inflammasome activation with IL-1β secretion in macrophages [45]. Thus, inflammation cytokines production by VVH appears to be different from that of other hemolysins.
The results of this study also provide additional insights into the relationship between TNF-α and ROS. Both TNF-α and ROS are crucial for inflammation, but the crosstalk between ROS and TNF-α during Infection is still not fully understood. The lectin-like domain of TNF-α has been reported to decrease LLO-induced Nox4 mRNA expression and ROS generation [46]. TNF-α activates NFκB signaling pathway in apoptotic cells and subsequently inhibits intracellular ROS level [23]. However, ROS generation is also induced by cytokines. In phagocytes, activation of the TNF-NOX2 signaling leads to ROS production responsible for inflammation and associated tissue damage [47]. Pore-forming toxin from Serratia marcescens can cause TNF-α-dependent necroptosis and facilitate ROS generation [48]. Thus, TNF is also important for ROS production. In this study, we showed that knockout TNF-α in murine macrophages does not affect ROS generation by low dose VVH treatment, which differs from the classic interconnection between TNF and ROS. Future work will focus on the mechanism underlying VVH-induced inflammation and oxidation stress.