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A critical role for hemolysin in Vibrio fluvialis-induced IL-1β secretion mediated by the NLRP3 inflammasome in macrophages.

Song L, Huang Y, Zhao M, Wang Z, Wang S, Sun H, Kan B, Meng G, Liang W, Ren Z - Front Microbiol (2015)

Bottom Line: The mice treated with 10(8) CFU wild-type V. fluvialis or cell-free supernatant containing VFH induced significantly higher IL-1β 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 has no effect on the synthesis of pro-IL-1β, but rather it triggers the processing of pro-IL-1β into IL-1β.Summary Sentence: Vibrio fluvialis-secreted hemolysin induces IL-1β secretion through the activation of the NLRP3 inflammasome and contributes to the pathogenicity of V. fluvialis.

View Article: PubMed Central - PubMed

Affiliation: State Key Laboratory for Infectious Disease Prevention and Control, Collaborative Innovation Center for Diagnosis and Treatment of Infectious Diseases, National Institute for Communicable Disease Control and Prevention - Chinese Center for Disease Control and Prevention Beijing, China.

ABSTRACT
Vibrio fluvialis causes human diarrhea, but the pathogenesis is not well-studied. We hypothesized that V. fluvialis-secreted hemolysin (VFH) may induce IL-1β 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. fluvialis-induced IL-1β 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 10(8) CFU wild-type V. fluvialis or cell-free supernatant containing VFH induced significantly higher IL-1β 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-1β release independent of colonization of V. fluvialis in the colon. VFH has no effect on the synthesis of pro-IL-1β, but rather it triggers the processing of pro-IL-1β into IL-1β. Furthermore, using deficient mouse strains, we verified that V. fluvialis-induced IL-1β 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-1β 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-1β secretion through the activation of the NLRP3 inflammasome and contributes to the pathogenicity of V. fluvialis.

No MeSH data available.


Related in: MedlinePlus

Vibrio fluvialis hemolysin-induced IL-1β production in BMMs requires ATP signaling, K+-efflux and ROS generation. (A,B) BMMs were infected with V. fluvialis WT or Δvfh in the absence or presence of KCl. IL-1β (A) and TNF-α (B) in the supernatants were determined at 3 h p.i. using ELISA. (C) The release of ATP from the BMMs infected with various strains was monitored using a bioluminescence assay kit. (D) The correlation between extracellular ATP release and IL-1β secretion is plotted for BMMs infected with different V. fluvialis strains at 3 h. (E–H) BMMs were infected with V. fluvialis WT or Δvfh in the absence or presence of oxidized ATP (oATP; E,F) or N-acetyl-L-cysteine (NAC; G,H). IL-1β and TNF-α in the supernatants were determined at 3 h p.i. using ELISA. Results represent mean ± SD of three independent experiments. ∗P < 0.05; ∗∗P < 0.01.
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Figure 7: Vibrio fluvialis hemolysin-induced IL-1β production in BMMs requires ATP signaling, K+-efflux and ROS generation. (A,B) BMMs were infected with V. fluvialis WT or Δvfh in the absence or presence of KCl. IL-1β (A) and TNF-α (B) in the supernatants were determined at 3 h p.i. using ELISA. (C) The release of ATP from the BMMs infected with various strains was monitored using a bioluminescence assay kit. (D) The correlation between extracellular ATP release and IL-1β secretion is plotted for BMMs infected with different V. fluvialis strains at 3 h. (E–H) BMMs were infected with V. fluvialis WT or Δvfh in the absence or presence of oxidized ATP (oATP; E,F) or N-acetyl-L-cysteine (NAC; G,H). IL-1β and TNF-α in the supernatants were determined at 3 h p.i. using ELISA. Results represent mean ± SD of three independent experiments. ∗P < 0.05; ∗∗P < 0.01.

Mentions: To investigate the role of K+ efflux in V. fluvialis-induced IL-1β production in BMMs, we added KCl to the cell culture medium to block K+ efflux prior to infection with V. fluvialis. KCl almost completely inhibited IL-1β production (Figure 7A), though no effects on TNF levels were observed (Figure 7B). Because extracellular ATP has been shown to trigger K+ efflux and NLRP3 activation via the ATP receptor P2X7 (Franchi et al., 2007), we sought to determine whether VFH affects extracellular ATP release. The results show that the WT and pUC-vfh strains induced significantly higher levels of ATP release into the supernatant than the Δvfh strain did (Figure 7C). Furthermore, there was a significantly positive correlation between ATP release and IL-1β secretion in BMMs (Figure 7D). To further study whether V. fluvialis exerts its effect through the ATP receptor P2X7, we tested the effects of the P2X7R inhibitor, oATP, on IL-1β release. oATP significantly reduced V. fluvialis-induced IL-1β levels (Figure 7E) although it had no effect on TNF-α production (Figure 7F). Finally, to determine the role of ROS in V. fluvialis-induced NLRP3 inflammasome activation, we pretreated BMMs with the ROS inhibitor NAC. NAC impaired V. fluvialis-induced IL-1β release in a dose-dependent manner (Figure 7G). NAC also reduced the production of TNF-α to a lesser extent (Figure 7H), which is different from the effects of KCl and oATP. Collectively, these findings suggest that V. fluvialis induction of IL-1β involves K+ efflux, extracellular ATP release, and the production of ROS.


A critical role for hemolysin in Vibrio fluvialis-induced IL-1β secretion mediated by the NLRP3 inflammasome in macrophages.

Song L, Huang Y, Zhao M, Wang Z, Wang S, Sun H, Kan B, Meng G, Liang W, Ren Z - Front Microbiol (2015)

Vibrio fluvialis hemolysin-induced IL-1β production in BMMs requires ATP signaling, K+-efflux and ROS generation. (A,B) BMMs were infected with V. fluvialis WT or Δvfh in the absence or presence of KCl. IL-1β (A) and TNF-α (B) in the supernatants were determined at 3 h p.i. using ELISA. (C) The release of ATP from the BMMs infected with various strains was monitored using a bioluminescence assay kit. (D) The correlation between extracellular ATP release and IL-1β secretion is plotted for BMMs infected with different V. fluvialis strains at 3 h. (E–H) BMMs were infected with V. fluvialis WT or Δvfh in the absence or presence of oxidized ATP (oATP; E,F) or N-acetyl-L-cysteine (NAC; G,H). IL-1β and TNF-α in the supernatants were determined at 3 h p.i. using ELISA. Results represent mean ± SD of three independent experiments. ∗P < 0.05; ∗∗P < 0.01.
© Copyright Policy - open-access
Related In: Results  -  Collection

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Show All Figures
getmorefigures.php?uid=PMC4440915&req=5

Figure 7: Vibrio fluvialis hemolysin-induced IL-1β production in BMMs requires ATP signaling, K+-efflux and ROS generation. (A,B) BMMs were infected with V. fluvialis WT or Δvfh in the absence or presence of KCl. IL-1β (A) and TNF-α (B) in the supernatants were determined at 3 h p.i. using ELISA. (C) The release of ATP from the BMMs infected with various strains was monitored using a bioluminescence assay kit. (D) The correlation between extracellular ATP release and IL-1β secretion is plotted for BMMs infected with different V. fluvialis strains at 3 h. (E–H) BMMs were infected with V. fluvialis WT or Δvfh in the absence or presence of oxidized ATP (oATP; E,F) or N-acetyl-L-cysteine (NAC; G,H). IL-1β and TNF-α in the supernatants were determined at 3 h p.i. using ELISA. Results represent mean ± SD of three independent experiments. ∗P < 0.05; ∗∗P < 0.01.
Mentions: To investigate the role of K+ efflux in V. fluvialis-induced IL-1β production in BMMs, we added KCl to the cell culture medium to block K+ efflux prior to infection with V. fluvialis. KCl almost completely inhibited IL-1β production (Figure 7A), though no effects on TNF levels were observed (Figure 7B). Because extracellular ATP has been shown to trigger K+ efflux and NLRP3 activation via the ATP receptor P2X7 (Franchi et al., 2007), we sought to determine whether VFH affects extracellular ATP release. The results show that the WT and pUC-vfh strains induced significantly higher levels of ATP release into the supernatant than the Δvfh strain did (Figure 7C). Furthermore, there was a significantly positive correlation between ATP release and IL-1β secretion in BMMs (Figure 7D). To further study whether V. fluvialis exerts its effect through the ATP receptor P2X7, we tested the effects of the P2X7R inhibitor, oATP, on IL-1β release. oATP significantly reduced V. fluvialis-induced IL-1β levels (Figure 7E) although it had no effect on TNF-α production (Figure 7F). Finally, to determine the role of ROS in V. fluvialis-induced NLRP3 inflammasome activation, we pretreated BMMs with the ROS inhibitor NAC. NAC impaired V. fluvialis-induced IL-1β release in a dose-dependent manner (Figure 7G). NAC also reduced the production of TNF-α to a lesser extent (Figure 7H), which is different from the effects of KCl and oATP. Collectively, these findings suggest that V. fluvialis induction of IL-1β involves K+ efflux, extracellular ATP release, and the production of ROS.

Bottom Line: The mice treated with 10(8) CFU wild-type V. fluvialis or cell-free supernatant containing VFH induced significantly higher IL-1β 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 has no effect on the synthesis of pro-IL-1β, but rather it triggers the processing of pro-IL-1β into IL-1β.Summary Sentence: Vibrio fluvialis-secreted hemolysin induces IL-1β secretion through the activation of the NLRP3 inflammasome and contributes to the pathogenicity of V. fluvialis.

View Article: PubMed Central - PubMed

Affiliation: State Key Laboratory for Infectious Disease Prevention and Control, Collaborative Innovation Center for Diagnosis and Treatment of Infectious Diseases, National Institute for Communicable Disease Control and Prevention - Chinese Center for Disease Control and Prevention Beijing, China.

ABSTRACT
Vibrio fluvialis causes human diarrhea, but the pathogenesis is not well-studied. We hypothesized that V. fluvialis-secreted hemolysin (VFH) may induce IL-1β 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. fluvialis-induced IL-1β 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 10(8) CFU wild-type V. fluvialis or cell-free supernatant containing VFH induced significantly higher IL-1β 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-1β release independent of colonization of V. fluvialis in the colon. VFH has no effect on the synthesis of pro-IL-1β, but rather it triggers the processing of pro-IL-1β into IL-1β. Furthermore, using deficient mouse strains, we verified that V. fluvialis-induced IL-1β 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-1β 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-1β secretion through the activation of the NLRP3 inflammasome and contributes to the pathogenicity of V. fluvialis.

No MeSH data available.


Related in: MedlinePlus