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Cecal Ligation and Puncture-induced Sepsis as a Model To Study Autophagy in Mice
Published on: February 9, 2014
Redox regulation of mitophagy in the lung during murine Staphylococcus aureus sepsis
Alan L Chang1, Allison Ulrich1, Hagir B Suliman2
1Department of Medicine, Duke University Medical Center, Durham, NC 27710, USA.
Abstract:
Oxidative mitochondrial damage is closely linked to inflammation and cell death, but low levels of reactive oxygen and nitrogen species serve as signals that involve mitochondrial repair and resolution of inflammation. More specifically, cytoprotection relies on the elimination of damaged mitochondria by selective autophagy (mitophagy) during mitochondrial quality control. This aim of this study was to identify and localize mitophagy in the mouse lung as a potentially upregulatable redox response to Staphylococcus aureus sepsis. Fibrin clots loaded with S. aureus (1×10(7) CFU) were implanted abdominally into anesthetized C57BL/6 and B6.129X1-Nfe2l2tm1Ywk/J (Nrf2(-/-)) mice. At the time of implantation, mice were given vancomycin (6mg/kg) and fluid resuscitation. Mouse lungs were harvested at 0, 6, 24, and 48h for bronchoalveolar lavage (BAL), Western blot analysis, and qRT-PCR. To localize mitochondria with autophagy protein LC3, we used lung immunofluorescence staining in LC3-GFP transgenic mice. In C57BL/6 mice, sepsis-induced pulmonary inflammation was detected by significant increases in mRNA for the inflammatory markers IL-1β and TNF-α at 6 and 24h, respectively. BAL cell count and protein also increased. Sepsis suppressed lung Beclin-1 protein, but not mRNA, suggesting activation of canonical autophagy. Notably sepsis also increased the LC3-II autophagosome marker, as well as the lung׳s noncanonical autophagy pathway as evidenced by loss of p62, a redox-regulated scaffolding protein of the autophagosome. In LC3-GFP mouse lungs, immunofluorescence staining showed colocalization of LC3-II to mitochondria, mainly in type 2 epithelium and alveolar macrophages. In contrast, marked accumulation of p62, as well as attenuation of LC3-II in Nrf2-knockout mice supported an overall decrease in autophagic turnover. The downregulation of canonical autophagy during sepsis may contribute to lung inflammation, whereas the switch to noncanonical autophagy selectively removes damaged mitochondria and accompanies tissue repair and cell survival. Furthermore, mitophagy in the alveolar region appears to depend on activation of Nrf2. Thus, efforts to promote mitophagy may be a useful therapeutic adjunct for acute lung injury in sepsis.
Insights
This study reveals that mitophagy, a cellular repair process, is activated in mouse lungs during Staphylococcus aureus sepsis. This process, dependent on Nrf2 activation, helps resolve inflammation and promotes tissue repair, offering a potential therapeutic target for acute lung injury.
Area of Science:
- Cell Biology
- Immunology
- Pathology
Background:
- Oxidative stress and inflammation are key in sepsis-induced cell death.
- Mitophagy, the selective removal of damaged mitochondria, is crucial for cellular quality control and resolving inflammation.
- Understanding mitophagy's role in sepsis is vital for developing new treatments.
Purpose of the Study:
- To identify and localize mitophagy in the mouse lung during Staphylococcus aureus sepsis.
- To investigate the role of the transcription factor Nrf2 in sepsis-induced mitophagy.
- To explore mitophagy as a potential therapeutic strategy for sepsis-related acute lung injury.
Main Methods:
- Induction of Staphylococcus aureus sepsis in C57BL/6 and Nrf2-knockout mice using S. aureus-loaded fibrin clots.
- Analysis of lung inflammation markers (IL-1β, TNF-α), bronchoalveolar lavage (BAL) fluid, and autophagy markers (Beclin-1, LC3-II, p62) via qRT-PCR and Western blot.
- Localization of mitophagy using immunofluorescence staining in LC3-GFP transgenic mice.
Main Results:
- Sepsis induced pulmonary inflammation and increased inflammatory markers (IL-1β, TNF-α) and BAL cell/protein counts.
- Sepsis altered autophagy markers, increasing LC3-II and decreasing p62, indicating activation of noncanonical autophagy.
- Mitophagy was localized to type 2 pneumocytes and alveolar macrophages, and its activation was dependent on Nrf2.
- Nrf2-knockout mice showed reduced autophagic turnover and increased p62 accumulation.
Conclusions:
- Downregulation of canonical autophagy may exacerbate lung inflammation during sepsis.
- A switch to noncanonical autophagy, including mitophagy, promotes mitochondrial quality control, tissue repair, and cell survival.
- Nrf2 activation is essential for mitophagy in the alveolar region during sepsis.
- Enhancing mitophagy could be a valuable therapeutic approach for acute lung injury in sepsis.
