Abstract
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.
Multiple organ failure syndrome is a highly lethal complication of severe sepsis lacking directed therapy, and involving poorly understood pathogenic mechanisms including mitochondrial damage. This mitochondrial damage is opposed by the induction of heme oxygenase-1 and by mitochondrial biogenesis.
In Staphylococcus aureus sepsis in mice, we demonstrate activation of mitochondrial biogenesis through a redox-regulated network involving the heme oxygenase-1/carbon monoxide system and the NF-E2–related factor-2 transcription factor, and modulated by Akt1, that promotes survival and is amenable to pharmacologic intervention.
Hospitalization rates for sepsis continue to rise, especially in the elderly (1), and gram-positive bacteria, particularly Staphylococcus aureus species, are responsible for most of the infections (2). Severe sepsis and septic shock cause death from multiple organ failure (MOF) syndrome, most notably when three or more organs fail (3, 4). The pathogenesis of MOF involves damage to mitochondria from immune effectors, such as tumor necrosis factor (TNF)-α (5), and by reactive oxygen species (ROS) and reactive nitrogen species that escape the antioxidant defenses (6). MOF has no specific therapy, but survival is associated with the induction of mitochondrial antioxidant defenses and mitochondrial biogenesis (7, 8).
The cell's oxidation-reduction (redox) state regulates the antioxidant defenses and broader adaptive genetic responses (9, 10) including those that regenerate mitochondria (11). The induction of mitochondrial biogenesis (12, 13) and the clearance of irreparably damaged organelles by mitophagy (14) are responsible for the maintenance of mitochondrial structure and function. The mitochondrial biogenesis program is also coupled to the antioxidant defense network (15).
The antioxidant enzyme heme oxygenase-1 (HO-1; Hmox-1) catalyzes the rate-limiting step in heme degradation (16). HO-1 induction protects against prooxidant heme release by many agents including LPS, cytokines, and ROS, whereas its products (iron, biliverdin, and carbon monoxide) have important physiologic effects (17, 18). Endogenous CO binds heme proteins, such as hemoglobin, cytochrome-c oxidase, and guanylate cyclase, affecting their functions (19, 20). CO binding to cytochrome oxidase increases mitochondrial ROS leakage, which promotes adaptive gene expression, including genes of mitochondrial biogenesis, through adjustments in redox state (21).
Hmox1 is thought to protect against sepsis-induced tissue damage, and it is induced by multiple transcriptional elements that respond to inflammation, especially the basic leucine zipper transcription factor, NF-E2 related factor-2 (Nrf2) (22). Nrf2 is normally sequestered in the cytosol by the cysteine-rich Kelch-like ECH-associated protein 1 (23), and Kelch-like ECH-associated protein 1 oxidation allows Nrf2 nuclear translocation (24) and binding to antioxidant response element (ARE) motifs located 5′ to the Hmox-1 transcription start site (25). Nrf2 also occupies activating ARE motifs in the nuclear respiratory factor-1 (NRF-1) promoter (15), and under the influence of CO, Nrf2 and NRF-1 along with NRF-2 (Gabpa) and the peroxisome proliferator-activated receptor gamma coactivator (PGC)-1 coactivators stimulate mitochondrial biogenesis (13, 26). Nrf2 also influences the innate immune response and survival in cecal ligation and puncture (27) and modulates leukocyte function in sepsis (28). However, the connection of these events to mitochondrial protection is unknown.
In addition, HO-1 is post-translationally regulated by Akt/PKB (29), and CO activates the kinase (30, 31). Akt phosphorylates NRF-1 and promotes its nuclear localization (32) and inactivates GSK3β, which improves Nrf2 nuclear translocation (33). These events form a basis for the role of HO-1 in the adaptive induction of mitochondrial biogenesis (15).
Endogenous CO contributes to the protective effects of HO-1 illustrated nicely by modulation of the antiinflammatory response (34) (i.e., through IL-10 [35], an antiinflammatory cytokine involved in LPS tolerance [36]). Mortality in rodents after LPS and after cecal ligation and puncture is reduced by CO or by CO-releasing molecules (37–39), and one study found an association between mitochondrial biogenesis and protection by CO (38). The link between Nrf2 and HO-1 suggests that CO could benefit sepsis resolution through Nrf2-mediated induction of mitochondrial biogenesis in concert with HO-1 and other prosurvival responses. We tested this hypothesis through intervention with CO in wild-type (WT) and Nrf2 and Akt1 null mice with S. aureus peritonitis. A portion of this work has been published in abstract form (40).
Antibodies were obtained from Santa Cruz, except for HO-1 (Assay Designs, Farmingdale, NY), PGC-1α (Cayman, Ann Arbor, MI), and phospho-Akt (S473 and T308; Cell Signaling, Danvers, MA). Polyclonal NRF-1, NRF-2 (Gabpa), PGC-1–related coactivator (PRC), and transcription factor-A (Tfam) were developed and characterized in our laboratory (11).
Studies were preapproved by our Institutional Animal Care and Use Committee. C57Bl6/J (WT) mice were obtained from Jackson Laboratory (Bar Harbor, ME). Nrf2−/− mice (Riken, Saitama, Japan) and Akt1−/− mice (Jackson) were bred institutionally and both sexes used at 9–15 weeks of age. S. aureus (clinical isolate Rosenbach Seattle 1945; ATCC [Manassas, VA] #25923; 1 × 106 to 108 cfu) was prepared, counted, and implanted abdominally in fibrin clots (7). Based on preliminary studies, we empirically administered CO at 250 ppm in air for 1 hour in breathing chambers on Days 1–3. This transiently increased carboxyhemoglobin level to approximately 20% without significant hypothermia or tissue hypoxia (21, 41). Some mice received HO inhibitor tin protoporphyrin (SnPP, 20 μmol/kg; Frontier Scientific) at surgery and again 24 hours later. Mice were killed at the indicated times and livers harvested for RNA, mtDNA, or mitochondrial or nuclear protein. Mitochondrial and nuclear pellets were stored in radioimmunoprecipitation assay buffer at −80°C.
Quantitative polymerase chain reaction was performed on an ABI PRISM 7000 with TaqMan expression assays (Applied Biosystems, Carlsbad, CA). 18S rRNA served as an endogenous control. Quantification of mRNA was determined by comparative threshold cycle CT and RQ method.
Protein was measured with bicinchoninic acid using bovine serum albumin standards. Fresh liver was hand-homogenized in standard isolation buffer and mitochondria isolated on 0.25% sucrose. Mitochondria were loaded onto a Rotofor (BioRad, Hercules, CA) containing an ampholyte (pH range, 3–10) and separated over 90 minutes by isoelectric focusing. For Western blotting, 10–20 μg protein were resolved by gradient sodium dodecyl sulfate–polyacrylamide gel electrophoresis (4–20%), transferred to Immobilon P, and blocked with 4% nonfat milk. Membranes were incubated at 4°C overnight with polyclonal anti–HO-1, p-Akt, Akt, porin, NRF-1, and Tfam (all 1:1,000); His3 (1:500); Nrf2 (1:800); PGC-1α (1:600); and PRC (1:200). The membranes were washed with Tris-buffered saline with Tween and incubated with horseradish peroxidase–conjugated secondary antibodies (Santa Cruz, Santa Cruz, CA); rewashed; and developed with enhanced chemiluminescence (Amersham, Piscataway, NJ). Digitized images were quantified in the middynamic range with BioRad ImageQuant. HO activity was measured in tissue and mitochondria using the CO reduction method (42).
Human HepG2 cells (ATCC) were cultured at 37°C in 5% CO2 in RMPI 1640 with 10% fetal calf serum, glutamine, and antibiotics. Near-confluent cells were exposed to peptidoglycan (PGN; 25 ng/ml) or dichloromethane (DCM/CO; 50 μM) to generate CO. Cells were transfected with scrambled or Nrf2 targeted siRNA using FuGene HD (Roche, Indianapolis, IN) to efficiencies and gene suppression of greater than 70% (43). Some cells were treated with DCM/CO and GSK-3β inhibitor TDZD-8 (25 μM; Calbiochem, St. Louis, MO) for 6 hours and some pretreated for 30 minutes with PI-3K/Akt inhibitor, Ly294002 (25 μM; Promega, Madison, WI).
Data for real-time reverse transcriptase polymerase chain reaction and protein densitometry are expressed as means ± SD for four to six samples. Significance was tested by Student unpaired t test or by two-way analysis of variance. Survival at 7 days was tested by Pearson chi square. Significance was set at P less than 0.05.
In C57BL/6J mice (WT), sepsis induced by implanting fibrin clots containing live S. aureus into the peritoneum followed by fluid resuscitation produces dose-dependent organ damage and lethality (7). Using 5 × 107 cfu S. aureus, we tested inhaled CO at 250 ppm for 1 hour at 2 hours after surgery, repeated daily for 3 days. In WT mice, 7-day mortality in the sepsis-control group was 48%, whereas mortality in the sepsis-CO treatment group was 23% (Figure 1A) (P < 0.05). CO was comparably protective at 1 × 108 cfu of S. aureus (not shown). We also performed sepsis studies on WT mice after HO inhibition and found them significantly more susceptible to mortality than sepsis-control mice (P < 0.05). They could not be rescued by CO (Figure 1A). We evaluated the liver histologically and found many inflammatory foci and areas of cell vacuolization and cell death in sepsis-control mice that were averted by the CO intervention (Figure 1B).
Figure 1. Staphylococcus aureus peritonitis and hepatic heme oxygenase-1 (HO-1; Hmox1) gene, HO-1 protein, and HO enzyme activity in wild-type (WT) mice. (A) Survival of WT sepsis-control or sepsis–carbon monoxide (CO) mice (arrows) after implantation of fibrin clots containing 5 × 107 cfu of S. aureus. Data are pooled from experiments of 12 mice per group done in duplicate. Inhaled CO is protective in S. aureus sepsis in WT mice (P = 0.014 by Pearson chi square); however, CO protection is lost when HO is inhibited with SnPP. (B) Histologic sections of the mouse liver stained with hematoxylin and eosin demonstrate increased inflammatory foci after S. aureus peritonitis and protection after intervention with CO. (C) Western analysis for HO-1 protein in sepsis-control and sepsis-CO mice showing increases in the hepatic protein level in sepsis-control and sepsis-CO mice (densitometry bars are mean ± SD for n = 4 per group and time; #P < 0.05 vs. time 0; *P < 0.05, vs. sepsis-control). (D) Real-time reverse transcriptase polymerase chain reaction for hepatic HO-1 mRNA in WT sepsis-control and sepsis-CO mice. HO-1 mRNA is significantly increased by CO. Bars are means ± SD of duplicates in three experiments (#P < 0.05 vs. time 0; *P < 0.05 vs. sepsis-control group at the same time point). (E) Hepatic HO enzyme activity in WT sepsis-control and sepsis-CO mice. HO activity peaks at 24 hours in sepsis-control and at 6 hours in sepsis-CO mice. Bars are mean ± SD of duplicates in three experiments (#P < 0.05 vs. time 0; *P < 0.05, vs. sepsis-control). (F) Distribution of liver mitochondrial HO activity (per microgram mitochondrial protein) as a function of mitochondrial pHi in control mice and after 24 hours in sepsis-control, sepsis-CO, and CO-only mice. HO enzyme activity was enriched in sepsis and highest in mitochondria at the extremes of pHi, implying activation of heme degradation in these subpopulations.
[More] [Minimize]To assess HO-1, we examined hepatic HO-1 protein, mRNA, and HO enzyme activity in C57BL/6J mice at 0, 6, 24, and 48 hours after implantation of 5 × 107 cfu S. aureus with or without CO. HO-1 protein and Hmox1 gene were up-regulated in sepsis-control mice at 6 and 24 hours, but sepsis-CO mice showed significantly greater Hmox1 up-regulation than sepsis-control mice (Figures 1C and 1D). CO also increased hepatic HO enzyme activity (Figure 1E), resulting in a higher CO production capacity and initiating a positive feedback cycle.
In healthy control mice (no sepsis), CO increased blood carboxyhemoglobin and tissue CO content commensurately with return to baseline within 2 hours (not shown); thus, we measured HO enzyme activity at 2 hours after CO breathing. In sepsis-control mice, HO activity gradually increased over 24 hours, whereas in sepsis-CO mice, HO activity increased within 6 hours and remained elevated.
We isolated liver mitochondria and separated them into populations by isoelectric point, and found enrichment of HO activity in certain subpopulations. Figure 1F shows the distribution of HO activity in liver mitochondria of control mice and at 24 hours in sepsis-control, sepsis-CO, and CO-alone mice. Mitochondrial HO activity in the liver was enhanced at high and low pHi in sepsis and after CO breathing, implying activation of heme catabolism by these stresses in specific groups of organelles.
Staphylococcus aureus sepsis rapidly induced proinflammatory TNF-α (Figure 2A) and nitric oxide synthase-2 (NOS2) production (Figure 2B). TNF-α increases mitochondrial ROS production and mitochondrial oxidative stress (44), whereas the effect of NO varies. Excessive NO inhibits respiration and damages cells, whereas physiologic NO stimulates mitochondrial biogenesis (45). Here TNF-α and NOS2 induction were decreased in sepsis-CO mice. Sepsis-control mice showed an oxidative stress response indicated by Nrf2 (Figure 2C) and superoxide dismutase 2 (Figure 2D) gene induction that was enhanced by CO. Sepsis-control mice also showed robust increases in antiinflammatory IL-10 mRNA by 6 hours; this too was enhanced by CO (Figure 2E).
Figure 2. Hepatic tumor necrosis factor (TNF)-α, nitric oxide synthase (NOS)-2, IL-10, and NF-E2–related factor-2 (Nrf2) antioxidant responses by real-time reverse transcriptase polymerase chain reaction during Staphylococcus aureus sepsis. (A) TNF-α mRNA up-regulation in sepsis-control mice is attenuated in sepsis–carbon monoxide (CO) mice (*P < 0.05 vs. time 0; #P < 0.05 vs. time 0 and sepsis-CO). (B) NOS2 mRNA is induced in sepsis-control mice by 6 hours, but is limited by inhaled CO (A and B: *P < 0.05 vs. time 0; #P < 0.05 vs. time 0 and with CO). (C) Nrf2 mRNA by real-time reverse transcriptase polymerase chain reaction is increased in sepsis-control mice by 6 hours, and expression of the gene is enhanced by CO inhalation. (D) Increased superoxide dismutase 2 mRNA levels in sepsis-control mice are further increased in sepsis-CO mice. (E) Increased IL-10 mRNA levels in sepsis-control mice are further increased in sepsis-CO mice (C, D, and E: *P < 0.05 vs. time 0; #P < 0.05 vs. time 0 and sepsis-control mice).
[More] [Minimize]During S. aureus sepsis in mice, aerobic function depends on mitochondrial replacement, which in turn depends on specific transcription factors, such as NRF-1 and NRF-2 (Gabpa), and coactivators, in particular PGC-1α, that up-regulate nuclear-encoded mitochondrial genes, such as Tfam, whose protein products are imported into mitochondria for mtDNA transcription and replication for mitochondrial biogenesis (46). In WT mice, hepatic NRF-1, Gabpa, PGC-1α, PRC, and Tfam mRNA levels increased in sepsis-control mice, but these effectors were further up-regulated in sepsis-CO mice (Figures 3A–3E). Sepsis-control mice confirmed a postinoculation fall in hepatic mtDNA copy number, which was abrogated by CO (Figure 3F).
Figure 3. Transcriptional up-regulation of mitochondrial biogenesis and protection of mtDNA copy number in Staphylococcus aureus sepsis. (A) Hepatic nuclear respiratory factor (NRF)-1 expression by real-time reverse transcriptase polymerase chain reaction increases in sepsis-control mice, reaching a maximum at 24 hours, with further increases in sepsis–carbon monoxide (CO) mice. (B) Hepatic up-regulation of Gabpa (NRF-2α) mRNA in sepsis-control mice parallels NRF-1 and further increases in sepsis-CO mice. (C) The peroxisome proliferator-activated receptor gamma coactivator (PGC)-1α coactivator is up-regulated by 6 hours of sepsis and further increases in sepsis-CO mice. (D) The PGC-1 family member PGC-1–related coactivator increases in sepsis-control mice and further increases in sepsis-CO mice. (E) Hepatic transcription factor-A (Tfam) mRNA level increases in sepsis-control mice and increases further in sepsis-CO mice. (A–E: #P < 0.05 vs. time 0; *P < 0.05 vs. time 0 and sepsis-control for n = 4 per time per group). (F) Log hepatic mitochondrial DNA copy number in sepsis-control and sepsis-CO mice. Inhaled CO reverses the decline in mtDNA copy number in sepsis (n = 4 per time per group; #P < 0.05 less than time 0 control; *P < 0.05 higher than the respective sepsis-control group).
[More] [Minimize]Nuclear NRF-1 and PGC-1α protein levels increased in sepsis-control, but this response occurred earlier in sepsis-CO mice (Figure 4A). Nuclear Gabpa and Nrf2 protein levels increased in sepsis-control mice with further up-regulation of these two proteins in sepsis-CO mice (Figure 4B). Mitochondrial ROS strongly influence the cellular oxidant–antioxidant balance, also involved in redox signaling (47). Overwhelming ROS production damages mitochondria, but oxidative stress stimulates Nrf2 translocation and binding to AREs in protective phase II antioxidant genes, including Hmox1 (48). HO-1/CO increases Nrf2 occupancy of the NRF1 promoter and increases NRF-1 production (15).
Figure 4. Up-regulation of mitochondrial biogenesis proteins during Staphylococcus aureus sepsis in wild-type mice by Western blot analysis. (A) Hepatic nuclear respiratory factor (NRF)-1 and peroxisome proliferator-activated receptor gamma coactivator-1α nuclear proteins increase in sepsis-control mice and increase further with carbon-monoxide (CO) (His3 is a nuclear reference protein). (B) Western analysis of hepatic Gabpa (NRF-2α) showing enhanced nuclear protein levels in sepsis-control and sepsis-CO mice. Nuclear NF-E2–related factor-2 (Nrf2) protein in sepsis-control and sepsis-CO mice is shown from the same nuclear samples compared with His3. For panels A and B, the densitometry confirms significant up-regulation of all four proteins by 24 hours in sepsis-control mice and further increases in sepsis-CO mice (for the densitometry, n = 4; #P < 0.05 vs. time 0; *P < 0.05 for the CO effect). (C) To verify the induction of mitochondrial biogenesis, we checked three mitochondrial marker proteins (nuclear-encoded citrate synthase [CS], mtDNA-encoded Complex I subunit NADH dehydrogenase 1, and the imported mitochondrial transcription factor transcription factor-A [Tfam]) (for the densitometry n = 4; # P < 0.05 above time 0 control; &P < 0.05 below time 0; *P < 0.05 for the CO effect). (D) Akt phosphorylation in sepsis-control mice is shown to increase at S473 and T308, but CO only enhances S473 phosphorylation (for densitometry, n = 4; #P < 0.05 vs. time 0; *P < 0.05 for the CO effect).
[More] [Minimize]To confirm the induction of mitochondrial biogenesis, we measured mitochondrial levels of the nuclear-encoded tricarboxylic acid cycle enzyme citrate synthase, the mitochondrial-encoded NADH dehydrogenase 1 (ND1) subunit of Complex I, and mitochondrial transcription factor Tfam (Figure 4C). There were losses in mitochondrial citrate synthase and ND1 induced by sepsis that were reversed earlier with CO than by the native responses. Tfam protein levels increased along with the activation of mitochondrial biogenesis (Figure 4C). CO promotes mitochondrial biogenesis through Akt and phosphorylation of NRF-1. In sepsis-control mice, hepatic Akt displayed greater phosphorylation at T308 and S473, but CO increased Akt phosphorylation only at S473, implying a relatively specific effect on Akt activation (Figure 4D).
Because of pronounced nuclear accumulation of Nrf2 in sepsis-control and sepsis-CO mice, we checked for overlapping mechanisms in HepG cells using S. aureus PGN and DCM/CO. PGN (25 ng/ml) increased the nuclear translocation of Nrf2 and that of NRF-1 (Figures 5A and 5B), and both effects were enhanced by DCM/CO (Figure 5C). Nrf2 was upstream of NRF-1 as demonstrated in Nrf2-silenced cells in which neither PGN nor DCM/CO, alone or in combination, stimulated the nuclear accumulation of NRF-1 (Figure 5D) (scrambled siRNA controls were negative, data not shown).
Figure 5. Nuclear NF-E2–related factor-2 (Nrf2) and nuclear respiratory factor (NRF)-1 accumulation after Staphylococcus aureus peptidoglycan (PGN) or dichloromethane (DCM)/carbon monoxide (CO) administration in HepG2 cells. (A) HepG2 cells were examined by confocal microscopy for the localization and expression of NRF-1 (red) and Nrf2 (green). Before PGN, both transcription factors are detectable only in the cytoplasm. Nuclear staining with Dapi (blue) is shown in the bottom panels of the columns. (B) After PGN exposure, both transcription factors accumulate in cell nuclei. (C) After exposure to PGN plus DCM/CO, intense homogeneous nuclear staining is noted for Nrf2 and NRF-1. (D) Nrf2 siRNA (siNrf2) blocks the effects of PGN plus DCM/CO on the nuclear accumulation of Nrf2 and NRF-1. Scrambled siRNA had no effect (no images shown).
[More] [Minimize]Sepsis mortality in Nrf2−/− mice was checked at a lower S. aureus inoculation dose of 5 × 106 cfu than in WT mice because Nrf2−/− mice were more susceptible to S. aureus mortality in preliminary studies. At the lower inoculation dose, Nrf2−/− sepsis-control mice died by 54 hours (Figure 6A). Moreover, all Nrf2−/− sepsis-CO mice died before the third CO treatment (implying toxicity). At 24 hours, the extent of hepatic congestion, cell damage, and inflammation in Nrf2−/− mice was more severe than in WT mice (Figure 6B). These findings support a survival role for Nrf2, a requirement for Nrf2 in CO protection, and the importance of early mitochondrial biogenesis in this S. aureus model.
Figure 6. NF-E2–related factor-2 (Nrf2) deletion and Staphylococcus aureus sepsis. (A) Nrf2−/− mice were implanted with fibrin clots containing S. aureus (5 × 106 cfu). The mice in the sepsis-control group all died by 54 hours and carbon monoxide (CO) offered no advantage (100% mortality by ∼ 36 h; P = NS; n = 12 mice per group). (B) Hematoxylin and eosin–stained liver sections in wild-type (WT) (left) and Nrf2−/− mice (right) at 0 and 24 hours after S. aureus inoculation (5 × 106 cfu). Extensive cell vacuolization, autolysis, and cell death are present in Nrf2−/− mouse liver. Horizontal arrows indicate inflammatory foci after sepsis, which are larger and more numerous in Nrf2−/− mice. Vertical arrows indicate foci of necrosis or apoptosis. (C) Heme oxygenase (HO)-1 mRNA levels in WT and Nrf2−/− mice. The WT response to CO is a positive control. In Nrf2−/− mice, S. aureus sepsis but not CO induces HO-1 mRNA expression (*P < 0.05 vs. sepsis-CO). (D) Hepatic HO-1 protein in Nrf2−/− mice by Western blot. S. aureus increases HO-1 protein, whereas CO does not. (E) Loss of hepatic mtDNA copy number in Nrf2−/− and Akt1−/− mice compared with WT mice at 24 hours of sepsis (*P < 0.05 compared with WT at time 0; **P < 0.05 compared with WT at 0 and 24 h). Note the higher S. aureus inoculation dose in WT mice. SA = S. aureus.
[More] [Minimize]For Hmox1 studies, separate Nrf2−/− mice were implanted with 5 × 106 S. aureus; half received two CO treatments and the livers harvested at approximately 28 hours. In Nrf2−/− mice, HO-1 mRNA increased approximately 2.5-fold in the sepsis-control group, but did not respond further in the sepsis-CO group (Figure 6C). Nrf2−/− sepsis-control mice also displayed normal hepatic HO-1 protein induction (Figure 6D); thus, Nrf2 was not the sole induction mechanism in S. aureus sepsis, but Nrf2 was required for CO to up-regulate HO-1. We assessed mtDNA copy number in the livers of the Nrf2−/− mice and in Akt1−/− mice and found a greater loss of mtDNA at 24 hours in Nrf2−/− compared with WT mice (Figure 6E).
Nrf2−/− mice in S. aureus sepsis did not induce mitochondrial biogenesis transcription factor genes normally; only Gabpa mRNA doubled, to half the WT levels (Figures 7A–7C). In Nrf2−/− mice, CO had no effect on NRF-1, Gabpa, or Tfam mRNA in sepsis. NRF-1 and PGC-1α protein increased in Nrf2−/− sepsis-control mice, but did not respond to CO (Figure 8A) and Tfam levels did not improve in Nrf2−/− sepsis-control mice or in Nrf2−/− sepsis-CO mice (Figure 8B). Akt phosphorylation increased in Nrf2−/− sepsis-control mice (Figure 8C), but CO had no effect, implying an interaction with Nrf2.
Figure 7. Transcriptional regulators of mitochondrial biogenesis in NF-E2–related factor-2 (Nrf2)−/− mice during Staphylococcus aureus sepsis. (A) Liver nuclear respiratory factor (NRF)-1 mRNA levels by real-time reverse transcriptase polymerase chain reaction increase less than twofold in Nrf2−/− sepsis-control mice and there is no significant response to CO. CO response in wild-type (WT) mice is provided as a positive control. (B) Gabpa mRNA levels in Nrf2−/− sepsis-control mice increase 2.5-fold, but there is no effect of CO. (C) Transcription factor-A (Tfam) mRNA expression does not increase in Nrf2−/− sepsis-control or sepsis-CO mice. Values are means ± SD of duplicates of three experiments (*P < 0.05 vs. baseline control). SA = S. aureus.
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Figure 8. Regulation of mitochondrial biogenesis regulatory proteins in NF-E2–related factor-2 (Nrf2)−/− mice during Staphylococcus aureus sepsis. (A) Western blots of total hepatic nuclear respiratory factor (NRF)-1 and PGC-1α protein showing minimal increases in Nrf2−/− sepsis-control mice, but none in carbon monoxide (CO) alone or in sepsis-CO mice (for densitometry, n = 4 per group). (B) Transcription factor-A (Tfam) is unaffected in Nrf2−/− sepsis-control, CO-only, or sepsis-CO mice (densitometry n = 4 per group). (C) In Nrf2−/− mice, Akt (S473) is phosphorylated in Nrf2−/− sepsis-control mice, but there are no effects of CO (densitometry n = 4 per group). SA = S. aureus.
[More] [Minimize]We exposed Akt1−/− mice to S. aureus at 5 × 106 cfu with or without CO intervention. In Akt1−/− sepsis-control mice, two-thirds of the group died by Day 7 (Figure 9A), and CO inhalation did not improve survival, indicating an Akt requirement for CO rescue (Figure 9A). Hmox1 induction was lost in Akt1−/− sepsis-control mice and CO actually interfered with HO-1 mRNA up-regulation (Figure 9B). Akt1−/− sepsis-control mice showed slight increases in hepatic HO-1 protein, but HO-1 did not respond to CO (Figure 9C). NRF-1 and Tfam protein increased slightly in Akt1−/− sepsis-control mice at 24 hours, but CO had no effect (Figure 9D). This implies impaired mitochondrial biogenesis connected to the absence of Akt phosphorylation of NRF-1 and the loss of Nrf2 regulation of NRF1.
Figure 9. Responses of Akt1−/− mice to Staphylococcus aureus sepsis. (A) Survival in Akt1−/− mice implanted with 5 × 106 cfu S. aureus was impaired compared with wild-type mice (Figure 1A) and did not improve with carbon monoxide (CO) administration. Seven-day mortality was 78% in sepsis-CO compared with 64% in sepsis-control mice (n = 12 per group; P = NS). (B) Real-time reverse transcriptase polymerase chain reaction for heme oxygenase (HO)-1 mRNA in Akt1−/− mice, unlike wild-type, shows no increase in sepsis. CO did not increase HO-1 mRNA in Akt1−/− mice, and mRNA levels are lower than sepsis-control mice at 24 and 48 hours (*P < 0.05 vs. CO). (C) Western blot showing increases in hepatic HO-1 protein levels in Akt1−/− sepsis-control mice at 24 hours, but CO had no effect on HO-1. (D) Hepatic protein levels for nuclear respiratory factor (NRF)-1 and transcription factor-A (Tfam) did not increase in Akt−/− sepsis-control or sepsis-CO mice. (E) Hepatic GSK3β phosphorylation increased in Akt−/− sepsis-control mice only at 6 hours, and CO had no effect. (F) HepG2 cells incubated with dichloromethane (DCM)/CO demonstrate the effect of CO on HO-1 protein expression. HO-1 induction by DCM/CO was blocked by inhibition of Akt and enhanced by inhibition of Gsk3β (densitometry n = 3; *P < 0.05 compared with lane 1).
[More] [Minimize]Nrf2 nuclear translocation is inhibited by Gsk3β, which is prevented by Akt (33), and we detected transient Gsk3β phosphorylation in Akt1−/− sepsis-control mice at 6 but not 24 hours. CO did not increase phosphoGsk3β levels, implying an Akt requirement for the inhibition of Gsk3β after CO (Figure 9E). This was checked in HepG2 cells, and the effects of DCM/CO on HO-1 were consistent with the mouse, and Akt inhibition blocked CO induction of HO-1, whereas Gsk3β inhibition enhanced it, indicating a critical role for Akt activation by CO in HO-1 induction (Figure 9F).
The resolution of mitochondrial damage after severe sepsis is fundamental to the recovery from sepsis-induced multiple organ dysfunction syndrome, a major cause of intensive care unit mortality. The mitochondrial quality control process in sepsis must be understood in the context of organ recovery to devise suitable mitochondrial therapy. The defense of mitochondria in sepsis is initiated by activation of innate immunity, which induces mitochondrial biogenesis through the Toll-like receptor system (43, 49, 50).
A role for the HO-1/CO system and the antiinflammatory effect of CO are well recognized, and despite potential CO toxicity, low doses of the gas can be administered without tissue hypoxia or cell damage (18). This presumably mimics the protective mechanisms of endogenous CO, and the affinity of CO for reduced heme proteins has implicated certain heme targets, including cytochrome oxidase, and physiologic CO stimulates mitochondrial biogenesis and leads to improved respiratory capacity (21). HO-1 is protective in sepsis, but the impact of the HO-1/CO system on mitochondrial integrity has remained ill-defined (38). To investigate this aspect, we tested whether pulse CO administration in mice would enhance HO-1 activity, thereby facilitating the induction of mitochondrial biogenesis and improving survival in S. aureus sepsis. A 1 hour per day CO regimen at 250 ppm for 3 days reduced 7-day sepsis mortality by half, and in a sentinel organ, the liver, induced Hmox1 through Nrf2 activation. The CO intervention expedited HO activity in mitochondria and the induction of mitochondrial biogenesis, promptly restoring mtDNA copy number. Moreover, Nrf2 stimulated antiinflammatory IL-10 expression, which dampens proinflammatory TNF-α and NOS2 production.
This mechanism was established by taking advantage of four facts. (1) Hmox1 is induced by Nrf2 (22). (2) HO-1/CO can activate mitochondrial biogenesis through Nrf2-inducible genes (15). (3) HO-1 is activated by and activates Akt (30, 51) and Akt phosphorylates NRF-1 during mitochondrial biogenesis (32). (4) HO-1 is linked to counter-inflammation, most discernibly through IL-10 (35), and coupling of counter-inflammation to mitochondrial biogenesis by Nrf2 (52).
HO-1 and its products lessen inflammatory tissue damage partly because increased heme catabolism decreases oxidative stress (17, 18, 53). In our S. aureus peritonitis model, this system reaches quasistability by 48 hours, but additional CO accelerated Hmox1 induction, leading to higher total and mitochondrial HO activity and a higher capacity for heme clearance. Lacking an obvious HO-1 importation mechanism, active enzyme associates with a subset of mitochondria to mitigate heme-induced oxidative stress.
In addition to Hmox1, Nrf2 activates other antioxidant and phase II detoxifying genes (54–56). Some Nrf2 is also associated with mitochondria (56), and Nrf2 activation is augmented by a surge in mitochondrial ROS generation after CO binding to cytochrome oxidase (15). Thus, HO-1 acting through CO generates a feed-forward cycle, apparently unique among Nrf2 network proteins, that meets the requirement for this transcription factor in mitochondrial biogenesis induction.
In fluid-resuscitated sepsis models, ROS produce sufficient damage to mitochondrial ultrastructure and function (57, 58) to be mitigated by antioxidant therapy (59, 60). In WT mice in sepsis, Nrf2 is a potent survival gene evidenced by a high sensitivity of Nrf2−/− mice to S. aureus lethality. Nrf2−/− sepsis-control mice show modest increases in hepatic HO-1 levels, but significantly less Hmox1 induction than WT mice. Thus, Hmox1 is induced by Nrf2-dependent and Nrf2-independent pathways in sepsis, but in Nrf2−/− mice, CO does not up-regulate HO-1, indicating an essential role for Nrf2 in CO protection. Accordingly, despite smaller S. aureus inoculations, CO fails to rescue Nrf2−/− mice. Moreover, when HO is inhibited, CO does not rescue WT mice, indicating a requirement for the Nrf2/HO-1 cycle. This also implies that the use of CO here does not depend on CO-dependent inhibition of S. aureus growth (39).
Nrf2 also up-regulates NRF1, and NRF-1 activates Tfam and other genes that control mtDNA transcription and replication. Oxidative mtDNA damage in sepsis is a result of innate immune activation, leading to a distinctive fall in hepatic mtDNA copy number (61), which is abrogated by CO intervention. Because Nrf2 regulates NRF-1 expression, we anticipated the weak induction of NRF-1 in Nrf2−/− sepsis-control mice, which along with disruption of the HO-1/CO cycle and suboptimal Gabpa and Tfam induction indicates impaired redox regulation of the mitochondrial biogenesis program. Moreover, failure of Nrf2−/− mice to activate mitochondrial biogenesis impedes counter-inflammation, which perpetuates mitochondrial damage caused by inflammatory cytokines, such as TNF-α (62).
Oxidative stress also activates Akt through the inactivation of specific phosphatases, such as phosphatase and tensin homolog, that counter-regulate PI-3K (62). CO enhances phosphatase inactivation through mitochondrial ROS production, leading to increased phosphorylation of Akt and NRF-1, which promotes mitochondrial biogenesis (32). Akt is phosphorylated by PI-3K at S473 and T308, and in WT and Nrf2−/− sepsis-control mice, Akt phosphorylation occurs at both sites, whereas CO enhances S473 phosphorylation. In Akt1−/− sepsis-control mice, Nrf2 activation is impaired by an unopposed inhibitory effect of GSK3β; Akt1−/− mice therefore lack NRF1 induction and cannot be rescued by CO.
Mechanistically, Nrf2 activation and Hmox1 expression improve survival in mice in S. aureus sepsis, presumably through a set of redox-activated Nrf2-dependent genes that support mitochondrial biogenesis. The pathways activated through this system are diagrammed in Figure 10. S. aureus and CO also both activate Akt, which acts upstream and directly through HO-1 on mitochondrial biogenesis, although we have an incomplete picture of its role. Nrf2−/− and Akt1−/− mice show increased sepsis mortality, and CO rescues WT but not mice lacking either of the two genes.
Figure 10. The heme oxygenase (HO)-1/carbon monoxide (CO) system and activation of mitochondrial biogenesis in Staphylococcus aureus sepsis. The diagram illustrates the redox cycle that activates NF-E2–related factor-2 (Nrf2) and the HO-1/CO system in response to S. aureus and to CO. Both the oxidative stress of innate immune hyperactivation and the CO intervention induce the mitochondrial biogenesis transcriptional program through Nrf2. The two known sites of Akt involvement are also indicated. Keap1 = Kelch-like ECH-associated protein 1; NF-κB = nuclear factor-κB; NRF1 = nuclear respiratory factor-1; PGC = peroxisome proliferator-activated receptor gamma coactivator; Tfam = transcription factor-A; ROS = reactive oxygen species; TLR = Toll-like receptor; TNF = tumor necrosis factor.
[More] [Minimize]We used CO to induce HO-1 through Nrf2, but other activators may also work, potentially with less toxicity, and CO may be less effective in lung injury in patients on high FiO2 because of low CO/O2 ratios. Thus, a novel sepsis survival mechanism involving mitochondrial heme sensing activates genes through Nrf2 that protect mtDNA and induce mitochondrial biogenesis. More specifically, HO-1, in the degradation of prooxidant heme, is the linchpin of an inducible mitochondrial quality control network that is transcriptionally integrated with the antioxidant and antiinflammatory defenses and amenable to pharmacologic exploitation.
The authors thank Craig Marshall, Marta Salinas, Lynn Tatro, John Patterson, and Kathy Stempel for excellent technical assistance.
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* Present address: Department of Medicine, Drexel College of Medicine, Philadelphia, PA 19102.
Supported by NIH RO1 AI064789, HL076528, and GM084116.
Author Contributions: N.C.M., H.B.S., and C.A.P., study conception and design; acquisition, interpretation, and analysis of data; drafting the article; and revising the article. R.R.B. and P.F., acquisition, analysis, and interpretation of data and revising the article. C.M.W., acquisition, analysis, interpretation of data and revising the article. K.E.W.-W., analysis and interpretation of data and revising the article. All authors approve of the final article.
Originally Published in Press as DOI: 10.1164/rccm.201106-1152OC on February 3, 2012
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