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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

Pseudomonas aeruginosapyocyanin activates the acid sphingomyelinase/ceramide system in mitochondria. (A–C) Mitochondria were isolated from wt or Asm-deficient neutrophils. Aliquots were stimulated with 50 μM pyocyanin for 10 or 30 min or with H2O2 for 10 min or left unstimulated. They were then lysed, and Asm activity (A, B) or mitochondrial ceramide concentrations (C) were determined. If indicated, 10 mM Tiron was added during the stimulations. Shown are the means±SD from four independent experiments; *p<0.05 compared with untreated samples, Δp<0.05 compared with treated wild-type cells, by ANOVA. (D, E) Wild-type or Asm-deficient peritoneal neutrophils were cultured on glass cover slips for 15 min, stimulated with 50 μM pyocyanin for 10 min, or left unstimulated. They were then fixed, permeabilized, stained with Cy3-coupled anti-ceramide and Cy5-coupled anti-Tim 23 antibodies, and analyzed by confocal microscopy. Shown are representative results from five independent experiments (D) and mean±SD (n=5) (E) of the fluorescence intensity in the Cy3 channel representing ceramide levels in mitochondria as defined by staining with Cy5-coupled anti-Tim antibodies. Fluorescence was quantified in at least 100 mitochondria per group, that is, 500 mitochondria total. The blue color represents Cy5, the red Cy3. *p<0.05 compared with untreated samples, Δp<0.05 compared with treated wild-type cells, ANOVA. H2O2, hydrogen peroxide; wt, wild-type.
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f5: Pseudomonas aeruginosapyocyanin activates the acid sphingomyelinase/ceramide system in mitochondria. (A–C) Mitochondria were isolated from wt or Asm-deficient neutrophils. Aliquots were stimulated with 50 μM pyocyanin for 10 or 30 min or with H2O2 for 10 min or left unstimulated. They were then lysed, and Asm activity (A, B) or mitochondrial ceramide concentrations (C) were determined. If indicated, 10 mM Tiron was added during the stimulations. Shown are the means±SD from four independent experiments; *p<0.05 compared with untreated samples, Δp<0.05 compared with treated wild-type cells, by ANOVA. (D, E) Wild-type or Asm-deficient peritoneal neutrophils were cultured on glass cover slips for 15 min, stimulated with 50 μM pyocyanin for 10 min, or left unstimulated. They were then fixed, permeabilized, stained with Cy3-coupled anti-ceramide and Cy5-coupled anti-Tim 23 antibodies, and analyzed by confocal microscopy. Shown are representative results from five independent experiments (D) and mean±SD (n=5) (E) of the fluorescence intensity in the Cy3 channel representing ceramide levels in mitochondria as defined by staining with Cy5-coupled anti-Tim antibodies. Fluorescence was quantified in at least 100 mitochondria per group, that is, 500 mitochondria total. The blue color represents Cy5, the red Cy3. *p<0.05 compared with untreated samples, Δp<0.05 compared with treated wild-type cells, ANOVA. H2O2, hydrogen peroxide; wt, wild-type.

Mentions: Next, we tested whether the Asm/ceramide system functions as a downstream target of pyocyanin and ROS, because oxygen radicals have been shown to activate Asm (27), which, in turn, releases ceramide, a proapoptotic molecule. To this end, we purified mitochondria from freshly isolated peritoneal neutrophils and determined the activity of Asm after treatment with pyocyanin or H2O2. The results (Fig. 5A, B) demonstrate a rapid activation of mitochondrial Asm activity by pyocyanin or H2O2. The addition of Tiron, an antioxidant, to isolated mitochondria prevented the activation of Asm by pyocyanin (Fig. 5A), a finding demonstrating the crucial role of ROS in Asm stimulation by pyocyanin in mitochondria.


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)

Pseudomonas aeruginosapyocyanin activates the acid sphingomyelinase/ceramide system in mitochondria. (A–C) Mitochondria were isolated from wt or Asm-deficient neutrophils. Aliquots were stimulated with 50 μM pyocyanin for 10 or 30 min or with H2O2 for 10 min or left unstimulated. They were then lysed, and Asm activity (A, B) or mitochondrial ceramide concentrations (C) were determined. If indicated, 10 mM Tiron was added during the stimulations. Shown are the means±SD from four independent experiments; *p<0.05 compared with untreated samples, Δp<0.05 compared with treated wild-type cells, by ANOVA. (D, E) Wild-type or Asm-deficient peritoneal neutrophils were cultured on glass cover slips for 15 min, stimulated with 50 μM pyocyanin for 10 min, or left unstimulated. They were then fixed, permeabilized, stained with Cy3-coupled anti-ceramide and Cy5-coupled anti-Tim 23 antibodies, and analyzed by confocal microscopy. Shown are representative results from five independent experiments (D) and mean±SD (n=5) (E) of the fluorescence intensity in the Cy3 channel representing ceramide levels in mitochondria as defined by staining with Cy5-coupled anti-Tim antibodies. Fluorescence was quantified in at least 100 mitochondria per group, that is, 500 mitochondria total. The blue color represents Cy5, the red Cy3. *p<0.05 compared with untreated samples, Δp<0.05 compared with treated wild-type cells, ANOVA. H2O2, hydrogen peroxide; wt, wild-type.
© Copyright Policy - open-access
Related In: Results  -  Collection

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getmorefigures.php?uid=PMC4403017&req=5

f5: Pseudomonas aeruginosapyocyanin activates the acid sphingomyelinase/ceramide system in mitochondria. (A–C) Mitochondria were isolated from wt or Asm-deficient neutrophils. Aliquots were stimulated with 50 μM pyocyanin for 10 or 30 min or with H2O2 for 10 min or left unstimulated. They were then lysed, and Asm activity (A, B) or mitochondrial ceramide concentrations (C) were determined. If indicated, 10 mM Tiron was added during the stimulations. Shown are the means±SD from four independent experiments; *p<0.05 compared with untreated samples, Δp<0.05 compared with treated wild-type cells, by ANOVA. (D, E) Wild-type or Asm-deficient peritoneal neutrophils were cultured on glass cover slips for 15 min, stimulated with 50 μM pyocyanin for 10 min, or left unstimulated. They were then fixed, permeabilized, stained with Cy3-coupled anti-ceramide and Cy5-coupled anti-Tim 23 antibodies, and analyzed by confocal microscopy. Shown are representative results from five independent experiments (D) and mean±SD (n=5) (E) of the fluorescence intensity in the Cy3 channel representing ceramide levels in mitochondria as defined by staining with Cy5-coupled anti-Tim antibodies. Fluorescence was quantified in at least 100 mitochondria per group, that is, 500 mitochondria total. The blue color represents Cy5, the red Cy3. *p<0.05 compared with untreated samples, Δp<0.05 compared with treated wild-type cells, ANOVA. H2O2, hydrogen peroxide; wt, wild-type.
Mentions: Next, we tested whether the Asm/ceramide system functions as a downstream target of pyocyanin and ROS, because oxygen radicals have been shown to activate Asm (27), which, in turn, releases ceramide, a proapoptotic molecule. To this end, we purified mitochondria from freshly isolated peritoneal neutrophils and determined the activity of Asm after treatment with pyocyanin or H2O2. The results (Fig. 5A, B) demonstrate a rapid activation of mitochondrial Asm activity by pyocyanin or H2O2. The addition of Tiron, an antioxidant, to isolated mitochondria prevented the activation of Asm by pyocyanin (Fig. 5A), a finding demonstrating the crucial role of ROS in Asm stimulation by pyocyanin in mitochondria.

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