Electronic cigarettes (E-cigs) have experienced sharp increases in popularity over the past five years due to many factors, including aggressive marketing, increased restrictions on conventional cigarettes, and a perception that E-cigs are healthy alternatives to cigarettes. Despite this perception, studies on health effects in humans are extremely limited and in vivo animal models have not been generated. Presently, we determined that E-cig vapor contains 7x1011 free radicals per puff. To determine whether E-cig exposure impacts pulmonary responses in mice, we developed an inhalation chamber for E-cig exposure. Mice that were exposed to E-cig vapor contained serum cotinine concentrations that are comparable to human E-cig users. E-cig exposure for 2 weeks produced a significant increase in oxidative stress and moderate macrophage-mediated inflammation. Since, COPD patients are susceptible to bacterial and viral infections, we tested effects of E-cigs on immune response. Mice that were exposed to E-cig vapor showed significantly impaired pulmonary bacterial clearance, compared to air-exposed mice, following an intranasal infection with Streptococcus pneumonia. This defective bacterial clearance was partially due to reduced phagocytosis by alveolar macrophages from E-cig exposed mice. In response to Influenza A virus infection, E-cig exposed mice displayed increased lung viral titers and enhanced virus-induced illness and mortality. In summary, this study reports a murine model of E-cig exposure and demonstrates that E-cig exposure elicits impaired pulmonary anti-microbial defenses. Hence, E-cig exposure as an alternative to cigarette smoking must be rigorously tested in users for their effects on immune response and susceptibility to bacterial and viral infections.
Although much is known about the pathophysiological processes contributing to diabetic retinopathy (DR), the role of protective pathways has received less attention. The transcription factor nuclear factor erythroid-2-related factor 2 (also known as NFE2L2 or NRF2) is an important regulator of oxidative stress and also has anti-inflammatory effects. The objective of this study was to explore the potential role of NRF2 as a protective mechanism in DR.
Retinal expression of NRF2 was investigated in human donor and mouse eyes by immunohistochemistry. The effect of NRF2 modulation on oxidative stress was studied in the human Müller cell line MIO-M1. Non-diabetic and streptozotocin-induced diabetic wild-type and Nrf2 knockout mice were evaluated for multiple DR endpoints.
NRF2 was expressed prominently in Müller glial cells and astrocytes in both human and mouse retinas. In cultured MIO-M1 cells, NRF2 inhibition significantly decreased antioxidant gene expression and exacerbated tert-butyl hydroperoxide- and hydrogen peroxide-induced oxidative stress. NRF2 activation strongly increased NRF2 target gene expression and suppressed oxidant-induced reactive oxygen species. Diabetic mice exhibited retinal NRF2 activation, indicated by nuclear translocation. Superoxide levels were significantly increased by diabetes in Nrf2 knockout mice as compared with wild-type mice. Diabetic Nrf2 knockout mice exhibited a reduction in retinal glutathione and an increase in TNF-α protein compared with wild-type mice. Nrf2 knockout mice exhibited early onset of blood–retina barrier dysfunction and exacerbation of neuronal dysfunction in diabetes.
These results indicate that NRF2 is an important protective mechanism regulating the progression of DR and suggest enhancement of the NRF2 pathway as a potential therapeutic strategy.
Diabetic retinopathy; Inflammation; Müller glial cells; Neuronal dysfunction; NF-E2-related factor-2; Reactive oxygen species; Transcription factor; Vascular permeability
Rtp801, a stress – related protein triggered by adverse environmental conditions, inhibits mTOR and enhances oxidative stress – dependent cell death. We postulated that Rtp801 acts as potential amplifying switch in the development of cigarette smoke – induced lung injury, leading to emphysema. Rtp801 was overexpressed in human emphysematous lungs and in lungs of mice exposed to cigarette smoke. The upregulation of Rtp801 expression by cigarette smoke in the lung relied on oxidative stress – dependent activation of the CCAAT response element. Rtp801 was necessary and sufficient for NF – κ B activation in cultured cells and, when forcefully expressed in mouse lungs, it promoted NF – kB activation, alveolar inflammation, oxidative stress, and apoptosis of alveolar septal cells. On the other hand, Rtp801 − / − mice were markedly protected against acute cigarette smoke – induced lung injury, partly via increased mTOR signaling, and, when exposed chronically, against emphysema. Our data support the notion that Rtp801 may represent an important molecular sensor and mediator of lung injury to cigarette smoke.
Rtp801; cigarette smoke; oxidative stress; apoptosis; inflammation; NF –κB; rapamycin
A nuclear disaster may result in exposure to potentially lethal doses of ionizing radiation (IR). Hematopoietic acute radiation syndrome (H-ARS) is characterized by severe myelosuppression, which increases the risk of infection, bleeding, and mortality. Here, we determined that activation of nuclear factor erythroid-2–related factor 2 (NRF2) signaling enhances hematopoietic stem progenitor cell (HSPC) function and mitigates IR-induced myelosuppression and mortality. Augmenting NRF2 signaling in mice, either by genetic deletion of the NRF2 inhibitor Keap1 or by pharmacological NRF2 activation with 2-trifluoromethyl-2′-methoxychalone (TMC), enhanced hematopoietic reconstitution following bone marrow transplantation (BMT). Strikingly, even 24 hours after lethal IR exposure, oral administration of TMC mitigated myelosuppression and mortality in mice. Furthermore, TMC administration to irradiated transgenic Notch reporter mice revealed activation of Notch signaling in HSPCs and enhanced HSPC expansion by increasing Jagged1 expression in BM stromal cells. Administration of a Notch inhibitor ablated the effects of TMC on hematopoietic reconstitution. Taken together, we identified a mechanism by which NRF2-mediated Notch signaling improves HSPC function and myelosuppression following IR exposure. Our data indicate that targeting this pathway may provide a countermeasure against the damaging effects of IR exposure.
Patients with COPD are associated with poor pulmonary anti-bacterial innate defenses, which increase the risk for frequent acute exacerbations caused by bacterial infection. Despite elevated numbers of phagocytes (macrophages and neutrophils), airways of patients with COPD show stable bacterial colonization. A defect in the phagocytic ability of alveolar macrophages (AMs) is one of the primary reasons for failure to clear the invading bacteria in airways of smokers and COPD patients and also in mice exposed to cigarette smoke (CS). Oxidative stress, as a result of CS exposure is implicated; however, the factors or mediators that inhibit phagocytic activity of AMs in lungs of smokers remain unclear. In the current study, we provide evidence that accumulation of oxidized phospholipids (Ox-PLs) mediate inhibition of phagocytic function of AMs in CS-exposed mice. Mice exposed to 6 months of CS showed impaired bacterial phagocytosis and clearance by AMs and elevated levels of Ox-PLs in bronchoalveolar lavage fluid (BALF), compared to mice exposed to room air. Intratracheal instillation of oxidized 1-palmitoyl-2-arachidonoyl-sn-glycero-3-phosphorylcholine (OX-PAPC) inhibited phagocytic activity of AMs and impaired pulmonary bacterial clearance in mice. In vitro studies demonstrated that exposure of J774 macrophages to OX-PAPC inhibited bacterial phagocytosis and clearance. However, pre-treatment of OX-PAPC with the monoclonal antibody EO6, which specifically binds to oxidized phospholipid but not native phospholipid, abolished OX-PAPC induced inhibition of bacterial phagocytosis and clearance. Incubation of BALF retrieved from CS-exposed mice impaired bacterial phagocytosis by J774 macrophages, which was abolished by pre-treatment of BALF with the EO6 antibody. In conclusion, our study shows that Ox-PLs generated following chronic CS exposure could play a crucial role in inhibiting phagocytic function of AMs and thus impair pulmonary anti-bacterial innate defenses in CS-exposed mice. Therapeutic approaches that augment pulmonary antioxidant defenses could be beneficial in reducing oxidative stress-driven impairment of phagocytosis by AMs in smokers and COPD patients.
COPD; macrophages; oxidized phospholipids; bacteria; phagocytosis; cigarette smoke
A growing body of evidence indicates that oxidative stress plays a central role in the progression of chronic obstructive pulmonary disease (COPD). Chronic oxidative stress caused by cigarette smoke generates damage-associated molecular patterns (DAMPs), such as oxidatively or nitrosatively modified proteins and extracellular matrix fragments, which induce abnormal airway inflammation by activating innate and adaptive immune responses. Furthermore, oxidative stress–induced histone deacetylase 2 (HDAC2) inactivity is implicated in amplifying inflammatory responses and corticosteroid resistance in COPD. Oxidative stress also mediates disruption of innate immune defenses, which is associated with acute exacerbation of COPD. Host defense transcription factor Nuclear factor erythroid 2–related factor 2 (Nrf2) regulates a multifaceted cytoprotective response to counteract oxidative stress–induced pathological injuries. A decrease in Nrf2 signaling is associated with the progression of diseases. Recent evidence indicates that targeting Nrf2 can be a novel therapy to mitigate inflammation, improve innate antibacterial defenses, and restore corticosteroid responses in patients with COPD.
COPD; Nrf2; bacteria; exacerbation; therapeutics
Rationale: Sepsis syndrome is characterized by inappropriate amplified systemic inflammatory response and bacteremia that promote multiorgan failure and mortality. Nuclear factor–erythroid 2 p45-related factor 2 (Nrf2) regulates a pleiotropic cytoprotective defense program including antioxidants and protects against several inflammatory disorders by inhibiting oxidative tissue injuries. However, the role of enhanced Nrf2 activity in modulating innate immune responses to microbial infection and pathogenesis of sepsis is unclear.
Objectives: To determine whether Nrf2 in myeloid leukocytes alters inflammatory response and protects against sepsis.
Methods: Mice with deletion of Nrf2 or kelch-like ECH-associated protein (Keap1) in myeloid leukocyte cells and respective floxed controls were subjected to cecal ligation and puncture–induced sepsis and were assessed for survival, organ injury, systemic inflammation, and bacteremia. Using LPS-stimulated peritoneal macrophages, Toll-like receptor (TLR) 4 surface trafficking and downstream signaling events were analyzed.
Measurements and Main Results: Mortality, organ injury, circulating levels of inflammatory mediators, and bacteremia were markedly reduced in LysM-Keap1−/− compared with respective floxed controls (Keap1f/f or Nrf2f/f) and significantly elevated in LysM-Nrf2−/− mice after cecal ligation and puncture. Peritoneal macrophages from septic LysM-Keap1−/− mice showed a greater bacterial phagocytic activity compared with LysM-Nrf2−/− and floxed controls. LPS stimulation resulted in greater reactive oxygen species–induced cell surface transport of TLR4 from trans-Golgi network and subsequent TLR4 downstream signaling (recruitment of MYD88 and TRIF, phosphorylation of IkB and IRF3, and cytokine expression) in macrophages of LysM-Nrf2−/− compared with LysM-Keap1−/− mice and floxed controls.
Conclusions: Our study shows that Nrf2 acts as a critical immunomodulator in leukocytes, controls host inflammatory response to bacterial infection, and protects against sepsis.
Nrf2; Keap1; sepsis; antioxidants; inflammation
Sulforaphane (SFN), an activator of nuclear factor erythroid-2 related factor 2 (Nrf2), is a promising chemopreventive agent which is undergoing clinical trial for several diseases. Studies have indicated that there is gain of Nrf2 function in lung cancer and other solid tumors because of mutations in the inhibitor Kelch-like ECH-associated protein 1 (Keap1). More recently, several oncogenes have been shown to activate Nrf2 signaling as the main prosurvival pathway mediating ROS detoxification, senescence evasion, and neoplastic transformation. Thus, it is important to determine if there is any risk of enhanced lung tumorigenesis associated with prolonged administration of SFN using mouse models of cancer.
Materials and Methods:
We evaluated the effect of prolonged SFN treatment on oncogenic K-ras (K-rasLSL-G12D)-driven lung tumorigenesis. One week post mutant-K-ras expression, mice were treated with SFN (0.5 mg, 5 d/wk) for 3 months by means of a nebulizer. Fourteen weeks after mutant K-ras expression (K-rasLSL-G12D), mice were sacrificed, and lung sections were screened for neoplastic foci. Expression of Nrf2-dependent genes was measured using real time RT-PCR. We also determined the effect of prolonged SFN treatment on the growth of preclinical xenograft models using human A549 (with mutant K-ras and Keap1 allele) and H1975 [with mutant epidermal growth factor receptor (EGFR) allele] nonsmall cell lung cancer cells.
Systemic SFN administration did not promote the growth of K-rasLSL-G12D-induced lung tumors and had no significant effect on the growth of A549 and H1975 established tumor xenografts in nude mice. Interestingly, localized delivery of SFN significantly attenuated the growth of A549 tumors in nude mice, suggesting an Nrf2-independent antitumorigenic activity of SFN.
Our results demonstrate that prolonged SFN treatment does not promote lung tumorigenesis in various mouse models of lung cancer.
EGFR; Keap1; K-ras; lung cancer; Nrf2; sulforaphane
Nrf2-mediated activation of antioxidant response element is a central part of molecular mechanisms governing the protective function of phase II detoxification and antioxidant enzymes against carcinogenesis, oxidative stress and inflammation. Nrf2 is sequestered in the cytoplasm by its repressor, Keap1. We have designed and synthesized novel chalcone derivatives as Nrf2 activators. The potency of these compounds was measured by the expression of Nrf2 dependent antioxidant genes, GCLM, NQO1 and HO1, in human lung epithelial cells; while the cytotoxicity was analyzed using MTT assay. In vivo potency of identified lead compounds to activate Nrf2 was evaluated using mouse model. Our studies showed 2-trifluoromethyl-2’-methoxychalone (2b) to be a potent activator of Nrf2, both, in vitro and in mice. Additional experiments showed that the activation of Nrf2 by this compound is independent of reactive oxygen species or redox changes. We have discussed a quantitative structure-activity relationship and proposed a possible mechanism of Nrf2 activation.
12-O-tetradecanoylphorbol 13-acetate (TPA) induces the differentiation of human myeloid ML-1 cells to macrophages. In the current study, the expression, responsiveness, and regulation of toll-like receptors (TLRs) in TPA-induced ML-1-derived macrophages were investigated. We have found that TPA-induced differentiation of ML-1 cells was accompanied by the upregulation of TLR1, TLR2, TLR4, and CD14 expression at both the mRNA and protein levels. Interestingly, TLR1 and TLR4 protein expression on ML-1 cells could be blocked by pretreatment with U0126, suggesting the role of an Erk1/2-induced differentiation signal in this process. In addition, the expression of IRAK-2, a key member of the TLR/IRAK-2/NF-κB-dependent signaling cascade was also induced in response to TPA. Accordingly, we demonstrated an increased cellular release of inflammatory cytokines (TNF-α and various interleukins) upon stimulation with LPS and LTA ligands for TLR4 and TLR2, respectively. Furthermore, using luminol-dependent chemiluminescence, addition of LPS and LTA induces a sustained DPI-inhibitable generation of reactive oxygen species (ROS) by the differentiated ML-1 cells. Together, these data suggest that the increase in the responsiveness of TPA-treated ML-1 cells to LPS and LTA occurs in response to the upregulation of their respective receptors as well as an induction of the IRAK-2 gene expression.
Chronic obstructive pulmonary disease (COPD), which is caused primarily by cigarette smoking, is a major health problem worldwide. The progressive decline in lung function that occurs in COPD is a result of persistent inflammation of the airways and destruction of the lung parenchyma. Despite the key role of inflammation in the pathogenesis of COPD, treatment with corticosteroids — normally highly effective antiinflammatory drugs — has little therapeutic benefit. This corticosteroid resistance is largely caused by inactivation of histone deacetylase 2 (HDAC2), which is critical for the transrepressive activity of the glucocorticoid receptor (GR) that mediates the antiinflammatory effect of corticosteroids. Here, we show that in alveolar macrophages from patients with COPD, S-nitrosylation of HDAC2 is increased and that this abolishes its GR-transrepression activity and promotes corticosteroid insensitivity. Cys-262 and Cys-274 of HDAC2 were found to be the targets of S-nitrosylation, and exogenous glutathione treatment of macrophages from individuals with COPD restored HDAC2 activity. Treatment with sulforaphane, a small-molecule activator of the transcription factor nuclear factor erythroid 2–related factor 2 (NRF2), was also able to denitrosylate HDAC2, restoring dexamethasone sensitivity in alveolar macrophages from patients with COPD. These effects of sulforaphane were glutathione dependent. We conclude that NRF2 is a novel drug target for reversing corticosteroid resistance in COPD and other corticosteroid-resistant inflammatory diseases.
Sepsis syndrome is characterized by a dysregulated inflammatory response to infection. NADPH oxidase-dependent reactive oxygen species (ROS) play significant roles in the pathophysiology of sepsis. We previously showed that disruption of Nrf2, a master regulator of antioxidant defenses, caused a dysregulation of innate immune response that resulted in greater mortality in a polymicrobial sepsis and lipopolysaccharide (LPS) shock model; however, the underlying mechanisms are unclear. In the present study, compared to wild-type (Nrf2+/+) macrophages, we observed greater PKC-induced NADPH oxidase-dependent ROS generation in Nrf2-disrupted (Nrf2−/−) macrophages that was modulated by glutathione (GSH) levels. To address the NADPH oxidase-mediated hyper-inflammatory response and sepsis-induced lung injury and mortality in Nrf2−/− mice, we used double knockout mice lacking Nrf2 and NADPH oxidase subunit, gp91phox (Nrf2−/−//Gp91phox−/−). Compared to Nrf2+/+ macrophages, LPS induced greater activation of TLR4 as evident by TLR4 surface trafficking and downstream recruitment of MYD88 and TRIF in Nrf2−/− macrophages that was diminished by ablation of gp91phox. Similarly, phosphorylation of IκB and IRF3 as well as cytokine expression was markedly higher in Nrf2−/− macrophages, while it was similar in Nrf2+/+ and Nrf2−/−//Gp91phox−/−. In vivo studies showed greater LPS-induced pulmonary inflammation in Nrf2−/− mice that was significantly reduced by ablation of gp91phox. Furthermore, LPS shock and polymicrobial sepsis induced early and greater mortality in Nrf2−/− mice, while Nrf2−/−//Gp91phox−/− showed prolong survival. Together, these results demonstrate that Nrf2 is essential for the regulation of NADPH oxidase-dependent ROS-mediated TLR4 activation and lethal innate immune response in sepsis.
Nuclear factor erythroid-2 related factor-2 (Nrf2) is a redox-sensitive transcription factor that regulates the expression of electrophile and xenobiotic detoxification enzymes and efflux proteins, which confer cytoprotection against oxidative stress and apoptosis in normal cells. Loss of function mutations in the Nrf2 inhibitor, Kelch-like ECH-associated protein (Keap1), results in constitutive activation of Nrf2 function in non-small-cell lung cancer (NSCLC). In this study, we demonstrate that constitutive activation of Nrf2 in lung cancer cells promotes tumorigenicity and contributes to chemoresistance by upregulation of glutathione, thioredoxin and the drug efflux pathways involved in detoxification of electrophiles and broad spectrum of drugs. RNAi-mediated reduction of Nrf2 expression in lung cancer cells induces generation of reactive oxygen species, suppresses tumor growth and results in increased sensitivity to chemotherapeutic drug induced cell death in vitro and in vivo. Inhibiting Nrf2 expression using naked siRNA duplexes in combination with carboplatin significantly inhibits tumor growth in a subcutaneous model of lung cancer. Thus, targeting Nrf2 activity in lung cancers, particularly those with Keap1 mutations, could be a promising strategy to inhibit tumor growth and circumvent chemoresistance.
Nrf2; Keap1; lung cancer; drug resistance; ROS; RNAi
► Nrf2 anti-oxidant function is impaired when HDAC activity is inhibited. ► HDAC inhibition decreases Nrf2 protein stability. ► HDAC2 is involved in reduced Nrf2 stability and both correlate in COPD samples. ► HDAC inhibition increases Nrf2 acetylation.
Nuclear factor erythroid 2-related factor 2 (Nrf2) plays a crucial role in cellular defence against oxidative stress by inducing the expression of multiple anti-oxidant genes. However, where high levels of oxidative stress are observed, such as chronic obstructive pulmonary disease (COPD), Nrf2 activity is reduced, although the molecular mechanism for this defect is uncertain. Here, we show that down-regulation of histone deacetylase (HDAC) 2 causes Nrf2 instability, resulting in reduced anti-oxidant gene expression and increase sensitivity to oxidative stress. Although Nrf2 protein was clearly stabilized after hydrogen peroxide (H2O2) stimulation in a bronchial epithelial cell line (BEAS2B), Nrf2 stability was decreased and Nrf2 acetylation increased in the presence of an HDAC inhibitor, trichostatin A (TSA). TSA also reduced Nrf2-regulated heme-oxygenase-1 (HO-1) expression in these cells, and this was confirmed in acute cigarette-smoke exposed mice in vivo. HDAC2 knock-down by RNA interference resulted in reduced H2O2-induced Nrf2 protein stability and activity in BEAS2B cells, whereas HDAC1 knockdown had no effect. Furthermore, monocyte-derived macrophages obtained from healthy volunteers (non-smokers and smokers) and COPD patients showed a significant correlation between HDAC2 expression and Nrf2 expression (r = 0.92, p < 0.0001). Thus, reduced HDAC2 activity in COPD may account for increased Nrf2 acetylation, reduced Nrf2 stability and impaired anti oxidant defences.
ARE, anti oxidant response element; COPD, chronic obstructive pulmonary disease; DJ-1, Parkinson’s disease (PD)-associated protein; HDAC2, histone deacetylase-2; HO-1, heme oxygenase-1; H2O2, hydrogen peroxide; Keap1, Kelch-like ECH associated protein 1; MDM, monocyte-derived macrophage; Nrf2, nuclear factor erythroid 2-related factor 2; ROS, reactive oxygen species; TSA, trichostatin A; Oxidative stress; Nrf2; Histone deacetylase 2; Protein stability; Acetylation; COPD
Rationale: Nuclear factor erythroid 2–related factor 2 (Nrf2), an important regulator of lung antioxidant defenses, declines in chronic obstructive pulmonary disease (COPD). However, Nrf2 also regulates the proteasome system that degrades damaged and misfolded proteins. Because accumulation of misfolded proteins in the endoplasmic reticulum (ER) causes ER stress and ER stress-induced apoptosis, Nrf2 may potentially prevent ER stress-mediated apoptosis in COPD.
Objectives: To determine whether Nrf2-regulated proteasome function affects ER stress-mediated apoptosis in COPD.
Methods: We assessed the expression of Nrf2, Nrf2-dependent proteasomal subunits, proteasomal activity, markers of ER stress, and apoptosis in emphysematous lungs of mice exposed to cigarette smoke (CS) as well as peripheral lung tissues from normal control subjects and patients with COPD.
Measurements and Main Results: Compared with wild-type mice, emphysematous lungs of CS-exposed Nrf2-deficient mice exhibited markedly lower proteasomal activity and elevated markers of ER stress and apoptosis. Furthermore, compared with normal control subjects, lungs of patients with mild and advanced COPD showed a marked decrease in the expression of Nrf2-regulated proteasomal subunits and total proteasomal activity. However, they were associated with greater levels of ER stress and apoptosis markers. In vitro studies have demonstrated that enhancing proteasomal activity in Beas2B cells either by sulforaphane, an activator of Nrf2, or overexpression of Nrf2-regulated proteasomal subunit PSMB6, significantly inhibited cigarette smoke condensate (CSC)-induced ER stress and cell death.
Conclusions: Impaired Nrf2 signaling causes significant decline in proteasomal activity and heightens ER stress response in lungs of patients with COPD and CS-exposed mice. Accordingly, pharmacological approaches that augment Nrf2 activity may protect against COPD progression by both up-regulating antioxidant defenses and relieving ER stress.
Nrf2; proteasome system; endoplasmic reticulum stress; unfolded protein response; chronic obstructive pulmonary disease lungs
Uveitis is an inflammatory condition that can lead to blindness. It is therefore, important to understand the pathophysiology on which to develop targeted therapy. Herein, we tested whether the oxidant-responsive transcription factor Nrf2 is involved in regulating the innate immune response and oxidative damage in the LPS uveitis model. With Dihydroethidium staining, intraperitoneally injected LPS increased reactive oxygen species in the retina and iris-ciliary body of Nrf2+/+ and Nrf2−/− mice. After LPS injection, ICAM-1, IL-6, TNF-α, COX-2, iNOS, and MCP-1 mRNA were increased more in the retina and iris-ciliary body of Nrf2−/− than Nrf2+/+ mice. NQO-1 and GCLM, two Nrf2 responsive anti-oxidant enzymes, had reduced expression in Nrf2+/+ retinas after LPS injection, but no change in expression in Nrf2−/− mice. The number of FITC-con A labeled leukocytes adherent to the retinal vascular endothelium increased after LPS treatment in both Nrf2+/+ and Nrf2−/− mice compared to control injections, with the more adherent leukocytes in Nrf2−/− than Nrf2+/+ mice. Pretreatment with the Nrf2 activator 1-(2-cyano-3-,12-dioxooleana-1,9(11)-dien-28-oyl)imidazole, increased antioxidant gene expression in the retina, reduced inflammatory mediator expression, and reduced leukocyte adherence to retinal vasculature after LPS treatment in Nrf2+/+ mice, but had no effect on Nrf2−/− mice. Treatment targeting the Nrf2 pathway may be new therapy for uveitis.
Adhesion molecules; Cytokines; Lipopolysaccharide (LPS); Nuclear factor erythroid-2 related factor 2 (Nrf2); Rodent; Transcription factors; Transgenic/knockout mice; triterpenoids; uveitis
Rationale: Oxidative stress is a key contributor in chronic obstructive pulmonary disease (COPD) pathogenesis caused by cigarette smoking. NRF2, a redox-sensitive transcription factor, dissociates from its inhibitor, KEAP1, to induce antioxidant expression that inhibits oxidative stress.
Objectives: To determine the link between severity of COPD, oxidative stress, and NRF2-dependent antioxidant levels in the peripheral lung tissue of patients with COPD.
Methods: We assessed the expression of NRF2, NRF2-dependent antioxidants, regulators of NRF2 activity, and oxidative damage in non-COPD (smokers and former smokers) and smoker COPD lungs (mild and advanced). Cigarette smoke–exposed human lung epithelial cells (Beas2B) and mice were used to understand the mechanisms.
Measurements and Main Results: When compared with non-COPD lungs, the COPD patient lungs showed (1) marked decline in NRF2-dependent antioxidants and glutathione levels, (2) increased oxidative stress markers, (3) significant decrease in NRF2 protein with no change in NRF2 mRNA levels, and (4) similar KEAP1 but significantly decreased DJ-1 levels (a protein that stabilizes NRF2 protein by impairing KEAP1-dependent proteasomal degradation of NRF2). Exposure of Bea2B cells to cigarette smoke caused oxidative modification and enhanced proteasomal degradation of DJ-1 protein. Disruption of DJ-1 in mouse lungs, mouse embryonic fibroblasts, and Beas2B cells lowered NRF2 protein stability and impaired antioxidant induction in response to cigarette smoke. Interestingly, targeting KEAP1 by siRNA or the small-molecule activator sulforaphane restored induction of NRF2-dependent antioxidants in DJ-1–disrupted cells in response to cigarette smoke.
Conclusions: NRF2-dependent antioxidants and DJ-1 expression was negatively associated with severity of COPD. Therapy directed toward enhancing NRF2-regulated antioxidants may be a novel strategy for attenuating the effects of oxidative stress in the pathogenesis of COPD.
chronic obstructive pulmonary disease; NRF2; DJ-1; oxidative stress; antioxidants
Extracellular signal-regulated kinase (Erk)1/2 activity signals myeloid cell differentiation induced by 12-O-tetradecanoyl-phorbol-13-acetate (TPA). Previously, we reported that Erk1/2 activation (phosphorylation) induced by TPA required reactive oxygen species (ROS) as a second messenger. Here, we hypothesized that ROS generated in response to TPA inhibit Erk1/2-directed phosphatase activity, which leads to an increase phosphorylation of Erk1/2 to signal p21WAF1/Cip1-mediated growth arrest in ML-1 cells. Incubation of ML-1 cells with TPA resulted in a marked accumulation of phosphorylated Erk1/2, and is subsequent to H2O2 generation. Interestingly, post-TPA-treatment with N-acetylcysteine (NAC) stimulated a marked and a rapid dephosphorylation of Erk1/2, suggesting a regeneration of Erk1/2-directed phospahatase activity by NAC. ROS generation in ML-1 cells induced by TPA was suggested to occur in the mitochondrial electron transport chain (METC) based on the following observations: (i) undifferentiated ML-1 cells not only lack p67-phox and but also express a low level of p47-phox key components required for NADPH oxidase enzymatic activity, (ii) pretreatment with DPI, an inhibitor of NADH- and NADPH-dependent enzymes, or rhein, an inhibitor of complex I, blocked the ROS generation, and (iii) examination of the microarray analysis data and Western blot analysis data revealed an induction of MnSOD expression at both mRNA and protein levels in response to TPA. MnSOD is a key member of the mitochondrial defense system against mitochondrial-derived superoxide. Together, this study suggested that TPA stimulated ROS generation as a second messenger to activate Erk1/2 via a redox-mediated inhibition of Erk1/2-directed phosphatase in ML-1 cells.
Oxidative stress and inflammation are two critical factors that drive the formation of plaques in atherosclerosis. Nrf2 is a redox-sensitive transcription factor that upregulates a battery of antioxidative genes and cytoprotective enzymes that constitute the cellular response to oxidative stress. Our previous studies have shown that disruption of Nrf2 in mice (Nrf2−/−) causes increased susceptibility to pulmonary emphysema, asthma and sepsis due to increased oxidative stress and inflammation. Here we have tested the hypothesis that disruption of Nrf2 in mice causes increased atherosclerosis.
To investigate the role of Nrf2 in the development of atherosclerosis, we crossed Nrf2−/− mice with apoliporotein E-deficient (ApoE−/−) mice. ApoE−/− and ApoE−/− Nrf2−/− mice were fed an atherogenic diet for 20 weeks, and plaque area was assessed in the aortas. Surprisingly, ApoE−/− Nrf2−/− mice exhibited significantly smaller plaque area than ApoE−/− controls (11.5% vs 29.5%). This decrease in plaque area observed in ApoE−/− Nrf2−/− mice was associated with a significant decrease in uptake of modified low density lipoproteins (AcLDL) by isolated macrophages from ApoE−/− Nrf2−/− mice. Furthermore, atherosclerotic plaques and isolated macrophages from ApoE−/− Nrf2−/− mice exhibited decreased expression of the scavenger receptor CD36.
Nrf2 is pro-atherogenic in mice, despite its antioxidative function. The net pro-atherogenic effect of Nrf2 may be mediated via positive regulation of CD36. Our data demonstrates that the potential effects of Nrf2-targeted therapies on cardiovascular disease need to be investigated.
The transcriptional factor Nrf2 has a unique role in various physiological stress conditions, but its contribution to ischemia/reperfusion injury has not been fully explored. Therefore, wildtype (WT) and Nrf2 knockout (Nrf2-/-) mice were subjected to 90-min occlusion of the middle cerebral artery (MCA) followed by 24-h reperfusion to elucidate Nrf2 contribution in protecting against ischemia/reperfusion injury. Infarct volume, represented as percent of hemispheric volume, was significantly (P<0.05) larger in Nrf2-/- mice than in WT mice (30.8 ± 6.1% vs 17.0 ± 5.1). Furthermore, neurological deficit was significantly greater in the Nrf2-/- mice. To examine whether neuronal protection was mediated by Nrf2, neurons were treated with various compounds to induce excitotoxic or oxidative stress. Translocation of Nrf2 into the nucleus was increased by the free-radical donor tert-butylhydroperoxide, but not by glutamate or NMDA. In addition, a common Nrf2 inducer, tert-butylhydroquinone, significantly attenuated neuronal cell death induced by tert-butylhydroperoxide (83.6 ± 1.6 vs 62.0 ± 7.7%) but not as substantially as when excitotoxicity was induced by NMDA (91.9 ± 1.6 vs 79.3 ± 3.3%) or glutamate (87.8 ± 1.5 vs 80.2 ± 2.6%). The results suggest that Nrf2 reduces ischemic brain injury by protecting against oxidative stress.
free radicals; MCA occlusion/reperfusion; NF-E2-related factor 2; stroke
Sepsis is characterized by an inappropriate host immune-inflammatory response and sustained oxidative damage. Nrf2, a bZIP oxidant-responsive transcription factor, regulates a battery of cytoprotective genes including antioxidants and maintains cellular redox homeostasis. Mouse studies have demonstrated a critical role of Nrf2 in improving survival during sepsis. This preclinical ex vivo study using neutrophils and peripheral blood mononuclear cells (PBMCs) as a surrogate cells evaluates the efficacy of CDDO-Im and CDDO-Me [imidazole and methyl ester derivative of 2-cyano-3,12-dioxooleana-1,9(11)-dien-28-oic acid (CDDO)] to activate the Nrf2 pathway and protect from lipopolysaccharide (LPS)-induced inflammatory response in humans. CDDO-Im treatment significantly induced Nrf2–dependent antioxidative genes (HO-1, GCLC, GCLM, and NQO1) in PBMCs isolated from six normal subjects. CDDO-Im increased nuclear accumulation of Nrf2 protein. Pretreatment of PBMC by CDDO-Im significantly attenuated LPS-induced cytokine expression. Similar increases in levels of antioxidant genes and suppression of LPS-induced cytokine expression was observed after CDDO-Me pretreatment. CDDO-Im also greatly inhibited LPS, fMLP, TNF-α, and TPA-induced ROS generation in neutrophils. In conclusion, these results demonstrate that activation of the Nrf2-dependent antioxidative pathway by CDDO-Im or CDDO-Me protects against the LPS-induced inflammatory response and suggest that they can be potential therapeutic candidates for intervening sepsis syndrome.
Sepsis induced lethality is characterized by amplified host innate immune response. Nrf2, a bZIP transcription factor regulates a battery of cellular antioxidative genes and maintains cellular redox homeostasis. This study demonstrates that increasing Nrf2 activity by a potent small molecule activator, CDDO-Im (1-[2-cyano-3-,12-dioxooleana-1,9(11)-dien-28-oyl]imidazole), protects from deregulation of lipopolysaccharide (LPS) induced innate immune response. In response to LPS stimuli, nrf2-deficient (nrf2 −/−) peritoneal neutrophils showed increased NADPH oxidase dependent ROS generation, proinflammatory cytokines (Tnf-α and Il-6) and chemokines (Mip2 and Mcp-1) relative to wild-type (nrf2 +/+) cells. Pretreatment of peritoneal neturophils with CDDO-Im induced antioxidative genes (Ho-1, Gclc, Gclm and Nqo1) and attenuated LPS induced ROS generation as well as expression of proinflammatory cytokines exclusively in nrf2 +/+ neutrophils but not in nrf2 −/− cells. In corroboration with in vitro studies, pretreatment with CDDO-Im induced Nrf2 dependent antioxidative genes, attenuated LPS induced proinflammatory cytokine expression and decreased septic shock induced mortality specifically in the nrf2 +/+ mice. In conclusion, the results suggest that Nrf2 is associated with oxidative regulation of LPS induced innate immune response in neutrophils. Activation of Nrf2 dependent compensatory antioxidative pathways by CDDO-Im protects from LPS induced inflammatory response and mortality due to septic shock.
Nrf2; CDDO-im; neutrophils; macrophages; innate immune response; antioxidant; ROS; LPS; Septic shock
Disruption of NF-E2–related factor (Nrf2), a redox-sensitive basic leucine zipper transcription factor, causes early-onset and more severe emphysema due to chronic cigarette smoke. Nrf2 determines the susceptibility of lungs to cigarette smoke–induced emphysema in mice through the transcriptional induction of numerous antioxidant genes. The lungs of Nrf2−/− mice have higher oxidative stress as evident from the increased levels of lipid peroxidation (4-hydroxy-2-nonenal) and oxidative DNA damage (7,8-dihydro-8-Oxo-2′deoxyguanosine) in response to cigarette smoke. Glutathione peroxidases (GPX) are the primary antioxidant enzymes that scavenge hydrogen peroxide and organic hydroperoxides. Among the five GPX isoforms, expression of GPX2 was significantly induced at both mRNA and protein levels in the lungs of Nrf2+/+ mice, in response to cigarette smoke. Activation of Nrf2 by specific knock down of the cytosolic inhibitor of Nrf2, Keap1, by small inhibitory RNA (siRNA) upregulated the expression of GPx2, whereas Nrf2 siRNA down-regulated the expression of GPX2 in lung epithelial cells. An ARE sequence located in the 5′ promoter–flanking region of exon 1 that is highly conserved between mouse, rat, and human was identified. Mutation of this ARE core sequence completely abolished the activity of promoter–reporter gene construct. The binding of Nrf2 to the GPX2 antioxidant response element was confirmed by chromatin immunoprecipation, electrophoretic mobility shift assays, and site-directed mutagenesis. This study shows that GPX2 is the major oxidative stress–inducible cellular GPX isoform in the lungs, and that its basal as well as inducible expression is dependent on Nrf2.
antioxidant response element; cigarette smoke; emphysema; GPX2; Nrf2
Nrf2 is a key transcriptional factor for antioxidant response element (ARE)-regulated genes. While its beneficial role has been described for stroke, its contribution to intracerebral hemorrhage (ICH)-induced early brain injury remains to be determined. Using wildtype (WT) and Nrf2 knockout (Nrf2−/−) mice, the role of Nrf2 in ICH induced by intracerebral injection of collagenase was investigated. The results showed that injury volume was significantly larger in Nrf2−/− mice than in WT controls 24 h after induction of ICH (p < 0.05), an outcome that correlated with neurological deficits. This exacerbation of brain injury in Nrf2−/− mice was also associated with an increase in leukocyte infiltration, production of reactive oxygen species, DNA damage, and cytochrome c release during the critical early phase of the post-ICH period. In combination, these results suggest that Nrf2 reduces ICH-induced early brain injury, possibly by providing protection against leukocyte-mediated free radical oxidative damage.
DNA damage; Free radicals; Inflammation; NF-E2-related factor 2; Reactive oxygen species