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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-secreted hemolysin-induced IL-1β secretion is dependent on Caspase-1 and NLRP3 inflammasome activation in mouse BMM. BMMs (1 × 106) were infected with WT, Δvfh, pUC-vfh, or pUC18 V. fluvialis strains at MOI 50 for 3 h or were incubated with PBS (negative control) or LPS + ATP (positive control). (A) Amounts of IL-1β p17 and p31 in supernatant (SN) and cell extract (CX) were visualized by Western blotting. (B) Amounts of active Caspase-1 p10 and procaspase-1 and in SN and CX were visualized by Western blotting. (C,D) The levels of IL-1β secretion (C) and TNF-α secretion (D) after infection of BMMs in the presence of the Caspase-1 inhibitor Z-YVAD-FMK were measured by ELISA. (E,F) The levels of IL-1β secretion (E) and TNF-α secretion (F) were measured in BMMs isolated from WT C57 B/6 or Caspase-1-/-, Nlrp3-/-, or Asc-/- mouse after infection with V. fluvialis. Values represent the mean + SD of triplicates and are representative of three independent experiments. ∗P < 0.05; ∗∗P < 0.01.
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Figure 5: Vibrio fluvialis-secreted hemolysin-induced IL-1β secretion is dependent on Caspase-1 and NLRP3 inflammasome activation in mouse BMM. BMMs (1 × 106) were infected with WT, Δvfh, pUC-vfh, or pUC18 V. fluvialis strains at MOI 50 for 3 h or were incubated with PBS (negative control) or LPS + ATP (positive control). (A) Amounts of IL-1β p17 and p31 in supernatant (SN) and cell extract (CX) were visualized by Western blotting. (B) Amounts of active Caspase-1 p10 and procaspase-1 and in SN and CX were visualized by Western blotting. (C,D) The levels of IL-1β secretion (C) and TNF-α secretion (D) after infection of BMMs in the presence of the Caspase-1 inhibitor Z-YVAD-FMK were measured by ELISA. (E,F) The levels of IL-1β secretion (E) and TNF-α secretion (F) were measured in BMMs isolated from WT C57 B/6 or Caspase-1-/-, Nlrp3-/-, or Asc-/- mouse after infection with V. fluvialis. Values represent the mean + SD of triplicates and are representative of three independent experiments. ∗P < 0.05; ∗∗P < 0.01.

Mentions: To study the mechanism of VFH-triggered IL-1β, we measured mRNA expression level of IL-1β induced by different strains and found that VFH had no effect on the mRNA expression of IL-1β (Supplementary Figure S1). We further examined the production of inactive pro-IL-1β (p31) and mature active IL-1β (p17) in supernatants and cell lysates using immunoblotting after infection. WT and pUC-vfh induced significantly higher levels of active mature IL-1β in the supernatants than Δvfh did, which is consistent with the ELISA results; however, all strains induced similar levels of biologically inactive pro-IL-1β in cell lysates (Figure 5A). Furthermore, the secretion of p10 subunit of Caspase-1 was evident in the supernatants of BMMs infected with WT and pUC-vfh but not in negative control cells or those treated with the Δvfh strain (Figure 5B). These results verify that the induction of IL-1β by VFH occurs at the level of inflammasome processing. To verify the role of Caspase-1, we repeated the ELISA experiments in the presence of the Caspase-1 inhibitor Z-YVAD-FMK. Z-YVAD-FMK dramatically inhibited IL-1β secretion (Figure 5C), but not TNF-α secretion (Figure 5D). Furthermore, V. fluvialis-induced IL-1β was abolished for BMMs isolated from Caspase-1-/-, Nlrp3-/-, or Asc-/- mice, though the release of TNF-α was not affected (Figures 5E,F). Giving the fact that VFH did not obviously affect message level of IL-1β and it indeed induced Caspase-1 activation and caused more mature IL-1β secreted, we conclude that VFH may affect IL-1β production at the level of IL-1β processing or secreting.


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-secreted hemolysin-induced IL-1β secretion is dependent on Caspase-1 and NLRP3 inflammasome activation in mouse BMM. BMMs (1 × 106) were infected with WT, Δvfh, pUC-vfh, or pUC18 V. fluvialis strains at MOI 50 for 3 h or were incubated with PBS (negative control) or LPS + ATP (positive control). (A) Amounts of IL-1β p17 and p31 in supernatant (SN) and cell extract (CX) were visualized by Western blotting. (B) Amounts of active Caspase-1 p10 and procaspase-1 and in SN and CX were visualized by Western blotting. (C,D) The levels of IL-1β secretion (C) and TNF-α secretion (D) after infection of BMMs in the presence of the Caspase-1 inhibitor Z-YVAD-FMK were measured by ELISA. (E,F) The levels of IL-1β secretion (E) and TNF-α secretion (F) were measured in BMMs isolated from WT C57 B/6 or Caspase-1-/-, Nlrp3-/-, or Asc-/- mouse after infection with V. fluvialis. Values represent the mean + SD of triplicates and are representative of three independent experiments. ∗P < 0.05; ∗∗P < 0.01.
© Copyright Policy - open-access
Related In: Results  -  Collection

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

Figure 5: Vibrio fluvialis-secreted hemolysin-induced IL-1β secretion is dependent on Caspase-1 and NLRP3 inflammasome activation in mouse BMM. BMMs (1 × 106) were infected with WT, Δvfh, pUC-vfh, or pUC18 V. fluvialis strains at MOI 50 for 3 h or were incubated with PBS (negative control) or LPS + ATP (positive control). (A) Amounts of IL-1β p17 and p31 in supernatant (SN) and cell extract (CX) were visualized by Western blotting. (B) Amounts of active Caspase-1 p10 and procaspase-1 and in SN and CX were visualized by Western blotting. (C,D) The levels of IL-1β secretion (C) and TNF-α secretion (D) after infection of BMMs in the presence of the Caspase-1 inhibitor Z-YVAD-FMK were measured by ELISA. (E,F) The levels of IL-1β secretion (E) and TNF-α secretion (F) were measured in BMMs isolated from WT C57 B/6 or Caspase-1-/-, Nlrp3-/-, or Asc-/- mouse after infection with V. fluvialis. Values represent the mean + SD of triplicates and are representative of three independent experiments. ∗P < 0.05; ∗∗P < 0.01.
Mentions: To study the mechanism of VFH-triggered IL-1β, we measured mRNA expression level of IL-1β induced by different strains and found that VFH had no effect on the mRNA expression of IL-1β (Supplementary Figure S1). We further examined the production of inactive pro-IL-1β (p31) and mature active IL-1β (p17) in supernatants and cell lysates using immunoblotting after infection. WT and pUC-vfh induced significantly higher levels of active mature IL-1β in the supernatants than Δvfh did, which is consistent with the ELISA results; however, all strains induced similar levels of biologically inactive pro-IL-1β in cell lysates (Figure 5A). Furthermore, the secretion of p10 subunit of Caspase-1 was evident in the supernatants of BMMs infected with WT and pUC-vfh but not in negative control cells or those treated with the Δvfh strain (Figure 5B). These results verify that the induction of IL-1β by VFH occurs at the level of inflammasome processing. To verify the role of Caspase-1, we repeated the ELISA experiments in the presence of the Caspase-1 inhibitor Z-YVAD-FMK. Z-YVAD-FMK dramatically inhibited IL-1β secretion (Figure 5C), but not TNF-α secretion (Figure 5D). Furthermore, V. fluvialis-induced IL-1β was abolished for BMMs isolated from Caspase-1-/-, Nlrp3-/-, or Asc-/- mice, though the release of TNF-α was not affected (Figures 5E,F). Giving the fact that VFH did not obviously affect message level of IL-1β and it indeed induced Caspase-1 activation and caused more mature IL-1β secreted, we conclude that VFH may affect IL-1β production at the level of IL-1β processing or secreting.

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