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Pseudomonas aeruginosa pyocyanin induces neutrophil death via mitochondrial reactive oxygen species and mitochondrial acid sphingomyelinase.

Managò A, Becker KA, Carpinteiro A, Wilker B, Soddemann M, Seitz AP, Edwards MJ, Grassmé H, Szabò I, Gulbins E - Antioxid. Redox Signal. (2015)

Bottom Line: Because many strains of P. aeruginosa are resistant to antibiotics, therapeutic options are limited.This reduced death, on the other hand, is associated with an increase in the release of interleukin-8 from pyocyanin-activated acid sphingomyelinase-deficient neutrophils but not from wild-type cells.These findings demonstrate a novel mechanism of pyocyanin-induced death of neutrophils and show how this apoptosis balances innate immune reactions.

View Article: PubMed Central - PubMed

Affiliation: 1 Department of Biology, University of Padova , Padova, Italy .

ABSTRACT

Aims: Pulmonary infections with Pseudomonas aeruginosa are a serious clinical problem and are often lethal. Because many strains of P. aeruginosa are resistant to antibiotics, therapeutic options are limited. Neutrophils play an important role in the host's early acute defense against pulmonary P. aeruginosa. Therefore, it is important to define the mechanisms by which P. aeruginosa interacts with host cells, particularly neutrophils.

Results: Here, we report that pyocyanin, a membrane-permeable pigment and toxin released by P. aeruginosa, induces the death of wild-type neutrophils; its interaction with the mitochondrial respiratory chain results in the release of reactive oxygen species (ROS), the activation of mitochondrial acid sphingomyelinase, the formation of mitochondrial ceramide, and the release of cytochrome c from mitochondria. A genetic deficiency in acid sphingomyelinase prevents both the activation of this pathway and pyocyanin-induced neutrophil death. This reduced death, on the other hand, is associated with an increase in the release of interleukin-8 from pyocyanin-activated acid sphingomyelinase-deficient neutrophils but not from wild-type cells.

Innovation: These studies identified the mechanisms by which pyocyanin induces the release of mitochondrial ROS and by which ROS induce neutrophil death via mitochondrial acid sphingomyelinase.

Conclusion: These findings demonstrate a novel mechanism of pyocyanin-induced death of neutrophils and show how this apoptosis balances innate immune reactions.

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Related in: MedlinePlus

Pyocyanin-induced cell death and mitochondrial cytochromecrelease require mitochondrial acid sphingomyelinase. (A, C) Peritoneal neutrophils or splenic neutrophils were obtained from wt or Asm-deficient mice. Cells were stimulated for 8 h with 50 μM pyocyanin, and apoptosis was analyzed by FITC-annexin V staining followed by flow cytometry. Shown are mean±SD, n=4; *p<0.05 compared with untreated, Δp<0.05 compared with treated wild-type cells, by ANOVA. (B) Wild-type or Asm-deficient mitochondria were incubated with 50 μM pyocyanin for 15 min and centrifuged. The pellets and the supernatants were subjected to Western blot analysis for cytochrome c. Cyochrome c in the pellet (P) of each sample represents mitochondrial cytochrome c; cytochrome c in the supernatant (S) of the same sample represents cyctochrome c released from mitochondria on induction of apoptosis. The sum equals total cytochrome c. Results are representative of six independent experiments.
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f7: Pyocyanin-induced cell death and mitochondrial cytochromecrelease require mitochondrial acid sphingomyelinase. (A, C) Peritoneal neutrophils or splenic neutrophils were obtained from wt or Asm-deficient mice. Cells were stimulated for 8 h with 50 μM pyocyanin, and apoptosis was analyzed by FITC-annexin V staining followed by flow cytometry. Shown are mean±SD, n=4; *p<0.05 compared with untreated, Δp<0.05 compared with treated wild-type cells, by ANOVA. (B) Wild-type or Asm-deficient mitochondria were incubated with 50 μM pyocyanin for 15 min and centrifuged. The pellets and the supernatants were subjected to Western blot analysis for cytochrome c. Cyochrome c in the pellet (P) of each sample represents mitochondrial cytochrome c; cytochrome c in the supernatant (S) of the same sample represents cyctochrome c released from mitochondria on induction of apoptosis. The sum equals total cytochrome c. Results are representative of six independent experiments.

Mentions: Pyocyanin-induced cell death in wild-type neutrophils was determined by fluorescein isothiocyanate (FITC)-annexin V staining of intact peritoneal neutrophils. Asm-deficient neutrophils resisted the toxin and exhibited significantly less apoptosis than wild-type neutrophils after treatment with pyocyanin (Fig. 7A). In accordance, only isolated mitochondria from wild-type peritoneal neutrophils, not those from Asm neutrophils, released cytochrome c after treatment with pyocyanin (Fig. 7B). Similar results were obtained with neutrophils isolated from the spleen and with HL-60 cells (Fig. 7C).


Pseudomonas aeruginosa pyocyanin induces neutrophil death via mitochondrial reactive oxygen species and mitochondrial acid sphingomyelinase.

Managò A, Becker KA, Carpinteiro A, Wilker B, Soddemann M, Seitz AP, Edwards MJ, Grassmé H, Szabò I, Gulbins E - Antioxid. Redox Signal. (2015)

Pyocyanin-induced cell death and mitochondrial cytochromecrelease require mitochondrial acid sphingomyelinase. (A, C) Peritoneal neutrophils or splenic neutrophils were obtained from wt or Asm-deficient mice. Cells were stimulated for 8 h with 50 μM pyocyanin, and apoptosis was analyzed by FITC-annexin V staining followed by flow cytometry. Shown are mean±SD, n=4; *p<0.05 compared with untreated, Δp<0.05 compared with treated wild-type cells, by ANOVA. (B) Wild-type or Asm-deficient mitochondria were incubated with 50 μM pyocyanin for 15 min and centrifuged. The pellets and the supernatants were subjected to Western blot analysis for cytochrome c. Cyochrome c in the pellet (P) of each sample represents mitochondrial cytochrome c; cytochrome c in the supernatant (S) of the same sample represents cyctochrome c released from mitochondria on induction of apoptosis. The sum equals total cytochrome c. Results are representative of six independent experiments.
© Copyright Policy - open-access
Related In: Results  -  Collection

Show All Figures
getmorefigures.php?uid=PMC4403017&req=5

f7: Pyocyanin-induced cell death and mitochondrial cytochromecrelease require mitochondrial acid sphingomyelinase. (A, C) Peritoneal neutrophils or splenic neutrophils were obtained from wt or Asm-deficient mice. Cells were stimulated for 8 h with 50 μM pyocyanin, and apoptosis was analyzed by FITC-annexin V staining followed by flow cytometry. Shown are mean±SD, n=4; *p<0.05 compared with untreated, Δp<0.05 compared with treated wild-type cells, by ANOVA. (B) Wild-type or Asm-deficient mitochondria were incubated with 50 μM pyocyanin for 15 min and centrifuged. The pellets and the supernatants were subjected to Western blot analysis for cytochrome c. Cyochrome c in the pellet (P) of each sample represents mitochondrial cytochrome c; cytochrome c in the supernatant (S) of the same sample represents cyctochrome c released from mitochondria on induction of apoptosis. The sum equals total cytochrome c. Results are representative of six independent experiments.
Mentions: Pyocyanin-induced cell death in wild-type neutrophils was determined by fluorescein isothiocyanate (FITC)-annexin V staining of intact peritoneal neutrophils. Asm-deficient neutrophils resisted the toxin and exhibited significantly less apoptosis than wild-type neutrophils after treatment with pyocyanin (Fig. 7A). In accordance, only isolated mitochondria from wild-type peritoneal neutrophils, not those from Asm neutrophils, released cytochrome c after treatment with pyocyanin (Fig. 7B). Similar results were obtained with neutrophils isolated from the spleen and with HL-60 cells (Fig. 7C).

Bottom Line: Because many strains of P. aeruginosa are resistant to antibiotics, therapeutic options are limited.This reduced death, on the other hand, is associated with an increase in the release of interleukin-8 from pyocyanin-activated acid sphingomyelinase-deficient neutrophils but not from wild-type cells.These findings demonstrate a novel mechanism of pyocyanin-induced death of neutrophils and show how this apoptosis balances innate immune reactions.

View Article: PubMed Central - PubMed

Affiliation: 1 Department of Biology, University of Padova , Padova, Italy .

ABSTRACT

Aims: Pulmonary infections with Pseudomonas aeruginosa are a serious clinical problem and are often lethal. Because many strains of P. aeruginosa are resistant to antibiotics, therapeutic options are limited. Neutrophils play an important role in the host's early acute defense against pulmonary P. aeruginosa. Therefore, it is important to define the mechanisms by which P. aeruginosa interacts with host cells, particularly neutrophils.

Results: Here, we report that pyocyanin, a membrane-permeable pigment and toxin released by P. aeruginosa, induces the death of wild-type neutrophils; its interaction with the mitochondrial respiratory chain results in the release of reactive oxygen species (ROS), the activation of mitochondrial acid sphingomyelinase, the formation of mitochondrial ceramide, and the release of cytochrome c from mitochondria. A genetic deficiency in acid sphingomyelinase prevents both the activation of this pathway and pyocyanin-induced neutrophil death. This reduced death, on the other hand, is associated with an increase in the release of interleukin-8 from pyocyanin-activated acid sphingomyelinase-deficient neutrophils but not from wild-type cells.

Innovation: These studies identified the mechanisms by which pyocyanin induces the release of mitochondrial ROS and by which ROS induce neutrophil death via mitochondrial acid sphingomyelinase.

Conclusion: These findings demonstrate a novel mechanism of pyocyanin-induced death of neutrophils and show how this apoptosis balances innate immune reactions.

Show MeSH
Related in: MedlinePlus