Elsevier

Free Radical Biology and Medicine

Volume 78, January 2015, Pages 179-189
Free Radical Biology and Medicine

Original Contribution
Redox regulation of mitophagy in the lung during murine Staphylococcus aureus sepsis

https://doi.org/10.1016/j.freeradbiomed.2014.10.582Get rights and content

Highlights

  • Proinflammatory cytokines cause increase in mitochondrial ROS production in response to S. aureus.

  • ROS-sensitive genes upregulate mitochondrial biogenesis.

  • Damaged mitochondria are disposed of by mitophagy.

  • Mitochondrial quality control is dependent on Nrf2.

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 (1×107 CFU) were implanted abdominally into anesthetized C57BL/6 and B6.129X1-Nfe2l2tm1Ywk/J (Nrf2−/−) mice. At the time of implantation, mice were given vancomycin (6 mg/kg) and fluid resuscitation. Mouse lungs were harvested at 0, 6, 24, and 48 h 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 24 h, 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.

Section snippets

Materials

Antibodies were purchased as follows: heme oxygenase 1 (HO-1; Assay Designs), Beclin-1 (Cell Signaling), Nrf2 (Santa Cruz), p62 (Abcam), LC3 (Sigma–Aldrich), SOD2 (Abcam), 8-hydroxy-2-deoxyguanosine (8-OHdG; Genetex). Primers used for quantitative real-time reverse transcriptase polymerase chain reaction (qRT-PCR) were all obtained from Life Technologies (Atg5, Atg12, Beclin-1, interleukin-1β (IL-1β), tumor necrosis factor-α (TNF-α), SOD2, HO-1). Bafilomycin A1 (BFA) was purchased from LC

Pulmonary inflammation and oxidative stress during sepsis

To assess the pulmonary inflammatory responses to S. aureus peritonitis, we measured lung mRNA levels for the early phase proinflammatory cytokines IL-1β and TNF-α at 0, 6, 24, and 48 h. The IL-1β and TNF-α mRNA levels increased sharply at 24 to 48 h after clot implantation (Figs. 1A and B). Cellular inflammation was also greatly increased as shown by significantly elevated bronchoalveolar lavage (BAL) white blood cell count and BAL protein at 24 h (Figs. 1C and D). To indirectly measure levels of

Discussion

Our data demonstrate for the first time that S. aureus sepsis induces mitophagy in the distal lung by a redox-sensitive pathway. Moreover, this redox pathway plays a key role in intracellular mitochondrial quality control, specifically in the turnover of damaged mitochondria as well as in mitochondrial biogenesis. In our mouse model, we found increased early phase inflammatory cytokine elaboration in the lung, i.e., IL-1β and TNF-α, as well as evidence of end organ inflammation and ALI

Acknowledgment

The authors thank the Duke Center for Hyperbaric Medicine and Environmental Physiology as well as the Eugene E. Stead Foundation for their generous scholarships and support.

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