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Mitochondria mediates caspase-dependent and independent retinal cell death in Staphylococcus aureus endophthalmitis

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ABSTRACT

Bacterial endophthalmitis, a vision-threatening complication of ocular surgery or trauma, is characterized by increased intraocular inflammation and retinal tissue damage. Although significant vision loss in endophthalmitis has been linked to retinal cell death, the underlying mechanisms of cell death remain elusive. In this study, using a mouse model of Staphylococcus aureus endophthalmitis and cultured human retinal Müller glia (MIO-M1 cell line), we demonstrate that S. aureus caused significant apoptotic cell death in the mouse retina and Müller glia, as evidenced by increased number of terminal dUTP nick end labeling and Annexin V and propidium iodide-positive cells. Immunohistochemistry and western blot studies revealed the reduction in mitochondrial membrane potential (JC-1 staining), release of cytochrome c into the cytosol, translocation of Bax to the mitochondria and the activation of caspase-9 and -3 in S. aureus-infected retina/retinal cells. In addition, the activation of PARP-1 and the release of apoptosis inducing factor from mitochondria was also observed in S. aureus-infected retinal cells. Inhibition studies using pan-caspase (Q-VD-OPH) and PARP-1 (DPQ) inhibitors showed significant reduction in S. aureus-induced retinal cell death both in vivo and in vitro. Together, our findings demonstrate that in bacterial endophthalmitis, retinal cells undergo apoptosis in the both caspase-dependent and independent manners, and mitochondria have a central role in this process. Hence, targeting the identified signaling pathways may provide the rationale to design therapeutic interventions to prevent bystander retinal tissue damage in bacterial endophthalmitis.

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S. aureus-challenged retinal Müller glia releases cytochrome c from mitochondria. MIO-M1 cells were left uninfected (control) or challenged with S. aureus (SA) RN6390 (multiplicity of infection, 10 : 1) for 8 h. Cells were stained with MitoTracker red (Mito Red) dye followed by immunostaining for cytochrome c and observed under confocal microscope (a). In another experiment, S. aureus-challenged MIO-M1 cells were subjected to subcellular fractionation followed by western blot analysis of cytochrome c (b). Cox 4 and β-actin antibodies were used as protein loading controls for mitochondrial and cytoplasmic fractions, respectively. Results are representative of two independent experiments.
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fig3: S. aureus-challenged retinal Müller glia releases cytochrome c from mitochondria. MIO-M1 cells were left uninfected (control) or challenged with S. aureus (SA) RN6390 (multiplicity of infection, 10 : 1) for 8 h. Cells were stained with MitoTracker red (Mito Red) dye followed by immunostaining for cytochrome c and observed under confocal microscope (a). In another experiment, S. aureus-challenged MIO-M1 cells were subjected to subcellular fractionation followed by western blot analysis of cytochrome c (b). Cox 4 and β-actin antibodies were used as protein loading controls for mitochondrial and cytoplasmic fractions, respectively. Results are representative of two independent experiments.

Mentions: Following mitochondrial membrane depolarization, the release of cytochrome c from the mitochondria, a fundamental event in apoptosis, initiates the assembly of the apoptosome resulting in activation of initiator caspase-9, the downstream effector caspases-3 and ultimately cell death.19,32 We, therefore, investigated the cellular distribution of cytochrome c using confocal imaging and subcellular fractionation studies. To this end, our data show the presence of cytochrome c in the cytoplasm in S. aureus-challenged Müller glia as observed by confocal imaging (Figure 3a). Furthermore, the subcellular fractionation and western blot analysis revealed increased cytochrome c levels in cytosolic fractions of infected cells as compared with control cells (Figure 3b).


Mitochondria mediates caspase-dependent and independent retinal cell death in Staphylococcus aureus endophthalmitis
S. aureus-challenged retinal Müller glia releases cytochrome c from mitochondria. MIO-M1 cells were left uninfected (control) or challenged with S. aureus (SA) RN6390 (multiplicity of infection, 10 : 1) for 8 h. Cells were stained with MitoTracker red (Mito Red) dye followed by immunostaining for cytochrome c and observed under confocal microscope (a). In another experiment, S. aureus-challenged MIO-M1 cells were subjected to subcellular fractionation followed by western blot analysis of cytochrome c (b). Cox 4 and β-actin antibodies were used as protein loading controls for mitochondrial and cytoplasmic fractions, respectively. Results are representative of two independent experiments.
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Related In: Results  -  Collection

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getmorefigures.php?uid=PMC4979429&req=5

fig3: S. aureus-challenged retinal Müller glia releases cytochrome c from mitochondria. MIO-M1 cells were left uninfected (control) or challenged with S. aureus (SA) RN6390 (multiplicity of infection, 10 : 1) for 8 h. Cells were stained with MitoTracker red (Mito Red) dye followed by immunostaining for cytochrome c and observed under confocal microscope (a). In another experiment, S. aureus-challenged MIO-M1 cells were subjected to subcellular fractionation followed by western blot analysis of cytochrome c (b). Cox 4 and β-actin antibodies were used as protein loading controls for mitochondrial and cytoplasmic fractions, respectively. Results are representative of two independent experiments.
Mentions: Following mitochondrial membrane depolarization, the release of cytochrome c from the mitochondria, a fundamental event in apoptosis, initiates the assembly of the apoptosome resulting in activation of initiator caspase-9, the downstream effector caspases-3 and ultimately cell death.19,32 We, therefore, investigated the cellular distribution of cytochrome c using confocal imaging and subcellular fractionation studies. To this end, our data show the presence of cytochrome c in the cytoplasm in S. aureus-challenged Müller glia as observed by confocal imaging (Figure 3a). Furthermore, the subcellular fractionation and western blot analysis revealed increased cytochrome c levels in cytosolic fractions of infected cells as compared with control cells (Figure 3b).

View Article: PubMed Central - PubMed

ABSTRACT

Bacterial endophthalmitis, a vision-threatening complication of ocular surgery or trauma, is characterized by increased intraocular inflammation and retinal tissue damage. Although significant vision loss in endophthalmitis has been linked to retinal cell death, the underlying mechanisms of cell death remain elusive. In this study, using a mouse model of Staphylococcus aureus endophthalmitis and cultured human retinal Müller glia (MIO-M1 cell line), we demonstrate that S. aureus caused significant apoptotic cell death in the mouse retina and Müller glia, as evidenced by increased number of terminal dUTP nick end labeling and Annexin V and propidium iodide-positive cells. Immunohistochemistry and western blot studies revealed the reduction in mitochondrial membrane potential (JC-1 staining), release of cytochrome c into the cytosol, translocation of Bax to the mitochondria and the activation of caspase-9 and -3 in S. aureus-infected retina/retinal cells. In addition, the activation of PARP-1 and the release of apoptosis inducing factor from mitochondria was also observed in S. aureus-infected retinal cells. Inhibition studies using pan-caspase (Q-VD-OPH) and PARP-1 (DPQ) inhibitors showed significant reduction in S. aureus-induced retinal cell death both in vivo and in vitro. Together, our findings demonstrate that in bacterial endophthalmitis, retinal cells undergo apoptosis in the both caspase-dependent and independent manners, and mitochondria have a central role in this process. Hence, targeting the identified signaling pathways may provide the rationale to design therapeutic interventions to prevent bystander retinal tissue damage in bacterial endophthalmitis.

No MeSH data available.


Related in: MedlinePlus