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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 induces release of AIF from mitochondria in retinal Müller glia. MIO-M1 cells were left uninfected (control) or challenged with S. aureus RN6390 (multiplicity of infection, 10 : 1) for the indicated time points. Cells were stained with MitoTracker red (Mito Red) followed by immunostaining for AIF and observed under confocal microscope (a). In another experiment, S. aureus-challenged MIO-M1 cells were subjected to subcellular fractionation followed by western blot for AIF (b). Cox 4 and β-actin antibodies were used as protein loading controls for mitochondrial and nuclear enriched fractions, respectively. Results are representative of two independent experiments.
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fig6: S. aureus induces release of AIF from mitochondria in retinal Müller glia. MIO-M1 cells were left uninfected (control) or challenged with S. aureus RN6390 (multiplicity of infection, 10 : 1) for the indicated time points. Cells were stained with MitoTracker red (Mito Red) followed by immunostaining for AIF and observed under confocal microscope (a). In another experiment, S. aureus-challenged MIO-M1 cells were subjected to subcellular fractionation followed by western blot for AIF (b). Cox 4 and β-actin antibodies were used as protein loading controls for mitochondrial and nuclear enriched fractions, respectively. Results are representative of two independent experiments.

Mentions: PARP-1 activation followed by the release of AIF from mitochondria is considered to be an important step for caspase-independent apoptotic cell death.33,34 We investigated the cellular localization of AIF in retinal Müller glia. Our data show that S. aureus challenge induces the nuclear localization of AIF, as revealed by immunostaining and confocal imaging (Figure 6a) as well as by western blot analysis of AIF in subcellular fractions (Figure 6b). These results suggest the existence of caspase-independent apoptotic mechanism in endophthalmitis, involving the activation of PARP-1 activation followed by nuclear localization of AIF.


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

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fig6: S. aureus induces release of AIF from mitochondria in retinal Müller glia. MIO-M1 cells were left uninfected (control) or challenged with S. aureus RN6390 (multiplicity of infection, 10 : 1) for the indicated time points. Cells were stained with MitoTracker red (Mito Red) followed by immunostaining for AIF and observed under confocal microscope (a). In another experiment, S. aureus-challenged MIO-M1 cells were subjected to subcellular fractionation followed by western blot for AIF (b). Cox 4 and β-actin antibodies were used as protein loading controls for mitochondrial and nuclear enriched fractions, respectively. Results are representative of two independent experiments.
Mentions: PARP-1 activation followed by the release of AIF from mitochondria is considered to be an important step for caspase-independent apoptotic cell death.33,34 We investigated the cellular localization of AIF in retinal Müller glia. Our data show that S. aureus challenge induces the nuclear localization of AIF, as revealed by immunostaining and confocal imaging (Figure 6a) as well as by western blot analysis of AIF in subcellular fractions (Figure 6b). These results suggest the existence of caspase-independent apoptotic mechanism in endophthalmitis, involving the activation of PARP-1 activation followed by nuclear localization of AIF.

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