Raquel R. Bartz

Heme Oxygenase-1 Couples Activation of Mitochondrial Biogenesis to Anti-inflammatory Cytokine Expression

The induction of heme oxygenase-1 (HO-1; Hmox1) by inflammation, for instance in sepsis, is associated both with an anti-inflammatory response and with mitochondrial biogenesis. Here, we tested the idea that HO-1, acting through the Nfe2l2 (Nrf2) transcription factor, links anti-inflammatory cytokine expression to activation of mitochondrial biogenesis. HO-1 induction after LPS stimulated anti-inflammatory IL-10 and IL-1 receptor antagonist (IL-1Ra) expression in mouse liver, human HepG2 cells, and mouse J774.1 macrophages but blunted tumor necrosis factor-α expression. This was accompanied by nuclear Nfe2l2 accumulation and led us to identify abundant Nfe2l2 and other mitochondrial biogenesis transcription factor binding sites in the promoter regions of IL10 and IL1Ra compared with pro-inflammatory genes regulated by NF-κΒ. Mechanistically, HO-1, through its CO product, enabled these transcription factors to bind the core IL10 and IL1Ra promoters, which for IL10 included Nfe2l2, nuclear respiratory factor (NRF)-2 (Gabpa), and MEF2, and for IL1Ra, included NRF-1 and MEF2. In cells, Hmox1 or Nfe2l2 RNA silencing prevented IL-10 and IL-1Ra up-regulation, and HO-1 induction failed post-LPS in Nfe2l2-silenced cells and post-sepsis in Nfe2l2−/− mice. Nfe2l2−/− mice compared with WT mice, showed more liver damage, higher mortality, and ineffective CO rescue in sepsis. Nfe2l2−/− mice in sepsis also generated higher hepatic TNF-α mRNA levels, lower NRF-1 and PGC-1α mRNA levels, and no enhancement of anti-inflammatory Il10, Socs3, or bcl-xL gene expression. These findings disclose a highly structured transcriptional network that couples mitochondrial biogenesis to counter-inflammation with major implications for immune suppression in sepsis.

Activation of Mitochondrial Biogenesis by Heme Oxygenase-1–mediated NF-E2–related Factor-2 Induction Rescues Mice from Lethal Staphylococcus aureus Sepsis

RATIONALE Mitochondrial damage is an important component of multiple organ failure syndrome, a highly lethal complication of severe sepsis that lacks specific therapy. Mitochondrial quality control is regulated in part by the heme oxygenase-1 (HO-1; Hmox1) system through the redox-regulated NF-E2-related factor-2 (Nrf2) transcription factor, but its role in mitochondrial biogenesis in Staphylococcus aureus sepsis is unknown. OBJECTIVES To test the hypothesis that Nrf2-dependent up-regulation of the HO-1/carbon monoxide (CO) system would preserve mitochondrial biogenesis and rescue mice from lethal S. aureus sepsis. METHODS A controlled murine S. aureus peritonitis model with and without inhaled CO was examined for HO-1 and Nrf2 regulation of mitochondrial biogenesis and the resolution of hepatic mitochondrial damage. MEASUREMENTS AND MAIN RESULTS Sepsis survival was significantly enhanced using inhaled CO (250 ppm once-daily for 1 h), and linked mechanistically to Hmox1 induction and mitochondrial HO activity through Nrf2 transcriptional and Akt kinase activity. HO-1/CO stimulated Nrf2-dependent gene expression and nuclear accumulation of nuclear respiratory factor-1, -2α (Gabpa), and peroxisome proliferator-activated receptor gamma coactivator-1α; increased mitochondrial transcription factor-A and citrate synthase protein levels; and augmented mtDNA copy number. CO enhanced antiinflammatory IL-10 and reduced proinflammatory tumor necrosis factor-α production. By contrast, Nrf2(-/-) and Akt1(-/-) mice lacked CO induction of Hmox1 and mitochondrial biogenesis, and CO rescued neither strain from S. aureus sepsis. CONCLUSIONS We identify an inducible Nrf2/HO-1 regulatory cycle for mitochondrial biogenesis that is prosurvival and counter-inflammatory in sepsis, and describe targeted induction of mitochondrial biogenesis as a potential multiple organ failure therapy.

Nrf2 promotes alveolar mitochondrial biogenesis and resolution of lung injury in Staphylococcus aureus pneumonia in mice

Acute lung injury (ALI) initiates protective responses involving genes downstream of the Nrf2 (Nfe2l2) transcription factor, including heme oxygenase-1 (HO-1), which stimulates mitochondrial biogenesis and related anti-inflammatory processes. We examined mitochondrial biogenesis during Staphylococcus aureus pneumonia in mice and the effect of Nrf2 deficiency on lung mitochondrial biogenesis and resolution of lung inflammation. S. aureus pneumonia established by nasal insufflation of live bacteria was studied in mitochondrial reporter (mt-COX8-GFP) mice, wild-type (WT) mice, and Nrf2⁻/⁻ mice. Bronchoalveolar lavage, wet/dry ratios, real-time RT-PCR and Western analysis, immunohistochemistry, and fluorescence microscopy were performed on the lung at 0, 6, 24, and 48 h. The mice survived S. aureus inoculations at 5×10⁸ CFU despite diffuse lung inflammation and edema, but the Nrf2⁻/⁻ lung showed increased ALI. In mt-COX8-GFP mice, mitochondrial fluorescence was enhanced in bronchial and alveolar type II (AT2) epithelial cells. WT mice displayed rapid HO-1 upregulation and lower proinflammatory TNF-α, IL-1β, and CCL2 and, especially in AT2 cells, higher anti-inflammatory IL-10 and suppressor of cytokine signaling-3 than Nrf2⁻/⁻ mice. In the alveolar region, WT but not Nrf2⁻/⁻ mice showed strongly induced nuclear respiratory factor-1, PGC-1α, mitochondrial transcription factor-A, SOD2, Bnip3, mtDNA copy number, and citrate synthase. These findings indicate that S. aureus pneumonia induces Nrf2-dependent mitochondrial biogenesis in the alveolar region, mainly in AT2 cells. Absence of Nrf2 suppresses the alveolar transcriptional network for mitochondrial biogenesis and anti-inflammation, which worsens ALI. The findings link redox activation of mitochondrial biogenesis to ALI resolution.

Pre-transplant mechanical ventilation and outcome in patients with cystic fibrosis.

Mechanical ventilation for ventilatory failure has been considered a relative contraindication to subsequent lung transplantation. The purpose of this study was to test the hypothesis that patients with cystic fibrosis (CF) who are intubated and mechanically ventilated before transplantation have poorer post-transplant outcomes than do patients who are not ventilated. We compared the outcomes of 8 patients with CF who underwent mechanical ventilation for 62 +/-20 days (range, 3-153 days) before bilateral lung transplantation with outcomes of 24 patients with CF who did not undergo pre-transplant mechanical ventilation. Although time to extubation after transplantation was prolonged significantly (11 vs 4 days) for the pre-transplant ventilated group, days to hospital discharge, forced expiratory volume in 1 second (percent predicted) at 1 year after transplantation, and post-transplant survival as determined using the Kaplan-Meier method did not differ statistically between the 2 groups. Patients with CF who undergo pre-transplant endotracheal intubation and mechanical ventilation for respiratory failure have outcomes that do not differ significantly from those of patients with CF who do not require invasive ventilatory support before bilateral lung transplantation.

Clinical review: Oxygen as a signaling molecule

Molecular oxygen is obviously essential for conserving energy in a form useable for aerobic life; however, its utilization comes at a cost - the production of reactive oxygen species (ROS). ROS can be highly damaging to a range of biological macromolecules, and in the past the overproduction of these short-lived molecules in a variety of disease states was thought to be exclusively toxic to cells and tissues such as the lung. Recent basic research, however, has indicated that ROS production - in particular, the production of hydrogen peroxide - plays an important role in both intracellular and extracellular signal transduction that involves diverse functions from vascular health to host defense. The present review summarizes oxygens capacity, acting through its reactive intermediates, to recruit the enzymatic antioxidant defenses, to stimulate cell repair processes, and to mitigate cellular damage.

Predicting Risk of Postoperative Lung Injury in High-risk Surgical Patients: A Multicenter Cohort Study

Background: Acute respiratory distress syndrome (ARDS) remains a serious postoperative complication. Although ARDS prevention is a priority, the inability to identify patients at risk for ARDS remains a barrier to progress. The authors tested and refined the previously reported surgical lung injury prediction (SLIP) model in a multicenter cohort of at-risk surgical patients. Methods: This is a secondary analysis of a multicenter, prospective cohort investigation evaluating high-risk patients undergoing surgery. Preoperative ARDS risk factors and risk modifiers were evaluated for inclusion in a parsimonious risk-prediction model. Multiple imputation and domain analysis were used to facilitate development of a refined model, designated SLIP-2. Area under the receiver operating characteristic curve and the Hosmer–Lemeshow goodness-of-fit test were used to assess model performance. Results: Among 1,562 at-risk patients, ARDS developed in 117 (7.5%). Nine independent predictors of ARDS were identified: sepsis, high-risk aortic vascular surgery, high-risk cardiac surgery, emergency surgery, cirrhosis, admission location other than home, increased respiratory rate (20 to 29 and ≥30 breaths/min), FIO2 greater than 35%, and SpO2 less than 95%. The original SLIP score performed poorly in this heterogeneous cohort with baseline risk factors for ARDS (area under the receiver operating characteristic curve [95% CI], 0.56 [0.50 to 0.62]). In contrast, SLIP-2 score performed well (area under the receiver operating characteristic curve [95% CI], 0.84 [0.81 to 0.88]). Internal validation indicated similar discrimination, with an area under the receiver operating characteristic curve of 0.84. Conclusions: In this multicenter cohort of patients at risk for ARDS, the SLIP-2 score outperformed the original SLIP score. If validated in an independent sample, this tool may help identify surgical patients at high risk for ARDS.

Airways obstruction in patients with sarcoidosis: expiratory CT scan findings.

In patients with pulmonary sarcoidosis, air trapping as evidenced by expiratory high-resolution computed tomography (HRCT) is not specific for a given stage of disease. Air trapping can occur at the level of the secondary lobule, as well as in distributions suggesting sublobular, subsegmental, and segmental involvement. While air trapping can be a nonspecific finding, it is a common feature in patients with pulmonary sarcoidosis and is a supportive diagnostic finding.

Staphylococcus aureus Sepsis and Mitochondrial Accrual of the 8-Oxoguanine DNA Glycosylase DNA Repair Enzyme in Mice

RATIONALE Damage to mitochondrial DNA (mtDNA) by the production of reactive oxygen species during inflammatory states, such as sepsis, is repaired by poorly understood mechanisms. OBJECTIVES To test the hypothesis that the DNA repair enzyme, 8-oxoguanine DNA glycosylase (OGG1), contributes to mtDNA repair in sepsis. METHODS Using a well-characterized mouse model of Staphylococcus aureus sepsis, we analyzed molecular markers for mitochondrial biogenesis and OGG1 translocation into liver mitochondria as well as OGG1 mRNA expression at 0, 24, 48, and 72 hours after infection. The effects of OGG1 RNA silencing on mtDNA content were determined in control, tumor necrosis factor-α, and peptidoglycan-exposed rat hepatoma cells. Based on in situ analysis of the OGG1 promoter region, chromatin immunoprecipitation assays were performed for nuclear respiratory factor (NRF)-1 and NRF-2α GA-binding protein (GABP) binding to the promoter of OGG1. MEASUREMENTS AND MAIN RESULTS Mice infected with 10(7) cfu S. aureus intraperitoneally demonstrated hepatic oxidative mtDNA damage and significantly lower hepatic mtDNA content as well as increased mitochondrial OGG1 protein and enzyme activity compared with control mice. The infection also caused increases in hepatic OGG1 transcript levels and NRF-1 and NRF-2α transcript and protein levels. A bioinformatics analysis of the Ogg1 gene locus identified several promoter sites containing NRF-1 and NRF-2α DNA binding motifs, and chromatin immunoprecipitation assays confirmed in situ binding of both transcription factors to the Ogg1 promoter within 24 hours of infection. CONCLUSIONS These studies identify OGG1 as an early mitochondrial response protein during sepsis under regulation by the NRF-1 and NRF-2α transcription factors that regulate mitochondrial biogenesis.

Redox mechanisms of cardiomyocyte mitochondrial protection

Oxidative and nitrosative stress are primary contributors to the loss of myocardial tissue in insults ranging from ischemia/reperfusion injury from coronary artery disease and heart transplantation to sepsis-induced myocardial dysfunction and drug-induced myocardial damage. This cell damage caused by oxidative and nitrosative stress leads to mitochondrial protein, DNA, and lipid modifications, which inhibits energy production and contractile function, potentially leading to cell necrosis and/or apoptosis. However, cardiomyocytes have evolved an elegant set of redox-sensitive mechanisms that respond to and contain oxidative and nitrosative damage. These responses include the rapid induction of antioxidant enzymes, mitochondrial DNA repair mechanisms, selective mitochondrial autophagy (mitophagy), and mitochondrial biogenesis. Coordinated cytoplasmic to nuclear cell-signaling and mitochondrial transcriptional responses to the presence of elevated cytoplasmic oxidant production, e.g., H2O2, allows nuclear translocation of the Nfe2l2 transcription factor and up-regulation of downstream cytoprotective genes such as heme oxygenase-1 which generates physiologic signals, such as CO that up-regulates Nfe212 gene transcription. Simultaneously, a number of other DNA binding transcription factors are expressed and/or activated under redox control, such as Nuclear Respiratory Factor-1 (NRF-1), and lead to the induction of genes involved in both intracellular and mitochondria-specific repair mechanisms. The same insults, particularly those related to vascular stress and inflammation also produce elevated levels of nitric oxide, which also has mitochondrial protein thiol-protective functions and induces mitochondrial biogenesis through cyclic GMP-dependent and perhaps other pathways. This brief review provides an overview of these pathways and interconnected cardiac repair mechanisms.

Peroxisome Proliferator-activated Receptor γ Co-activator 1-α as a Critical Co-activator of the Murine Hepatic Oxidative Stress Response and Mitochondrial Biogenesis in Staphylococcus aureus Sepsis

Background: PGC-1α regulates mitochondrial biogenesis, and may participate in antioxidant gene regulation. Results: PGC-1α-deficient mice in sepsis demonstrated increased hepatocellular mitochondrial oxidative stress and impaired antioxidant enzyme induction, reflecting PGC-1α interaction with the ARE-dependent Nfe2l2 transcription factor and Sod2 activation. Conclusion: PGC-1α is critical to mitochondrial SOD-2 induction during hepatic inflammation. Significance: This novel pathway offers unique opportunities to mitigate oxidative mitochondrial damage. A key transcriptional regulator of cell metabolism, the peroxisome proliferator-activated receptor γ co-activator 1-α (PPARGC-1-α or PGC-1α), also regulates mitochondrial biogenesis, but its role in antioxidant gene regulation is not well understood. Here, we asked whether genetic heterozygosity of PGC-1α modulates gene expression for the mitochondrial antioxidant enzyme SOD-2 during hepatic inflammatory stress. Using Staphylococcus aureus peritonitis in mice, we found significant Sod2 gene induction in WT mice, whereas PGC-1α heterozygotes (PGC-1α+/−) failed to augment Sod2 mRNA and protein levels. Impaired Sod2 regulation in PGC-1α+/− mice was accompanied by oxidative stress shown by elevated mitochondrial GSSG/GSH and protein carbonyls. In silico analysis of the mouse proximal Sod2 promoter region revealed consensus binding sites for the Nfe2l2 (Nrf2) transcription factor. Chromatin immunoprecipitation demonstrated diminished Nfe2l2 protein binding to the antioxidant response element promoter site proximal to the Sod2 start site in PGC-1α heterozygous mice, implicating PGC-1α in facilitation of Nfe2l2 DNA binding. Nuclear protein co-immunoprecipitation demonstrated an interaction between hepatic Nfe2l2 and PGC-1α in WT mice that was greatly reduced in PGC-1α+/− mice. The data indicate that PGC-1α promotes mitochondrial antioxidant enzyme expression through Nfe2l2-mediated SOD-2 expression in sepsis. The presence of this new PGC-1α-dependent signaling axis indicates that PGC-1α opposes mitochondrial oxidative stress by means of selective induction of one or more antioxidant response element-driven genes. By implication, exploitation of this axis could lead to new pharmacological interventions to improve the antioxidant defenses during oxidative stress-induced mitochondrial damage.