Nrf2 is a critical regulator of the innate immune response and survival during experimental sepsis (original) (raw)
Disruption of Nrf2 causes drastic increase in lethality during LPS and cecal ligation and puncture–induced septic shock. First, we examined the role of Nrf2 on the survival of wild-type (Nrf2+/+) and _Nrf2_-deficient (_Nrf2_–/–) mice during an endotoxic shock. Nrf2+/+ and _Nrf2_–/– mice were treated i.p. with a lethal dose of LPS (0.75 and 1.5 mg/mouse), and survival was monitored for 5 days. The lower dose resulted in the death of 50% of the _Nrf2_–/– mice but none of the Nrf2+/+ mice (Figure 1A). At the higher dose, 100% of the _Nrf2_–/– mice died within 48 hours whereas only 50% of the Nrf2+/+ mice died by day 5 (Figure 1B). Next, we investigated the role of Nrf2 on survival in a clinically relevant model of septic shock induced by cecal ligation and puncture (CLP). By 48 hours after CLP, all _Nrf2_–/– mice died while only 20% of wild-type littermates died. After 5 days, 40% of wild-type mice still survived (Figure 1C). No death was observed in sham-operated mice of both genotypes.
Nrf2–/– mice were more sensitive to LPS and septic peritonitis–induced septic shock. (A and B) Mortality after LPS administration. Age-matched male Nrf2+/+ (n = 10) and _Nrf2_–/– mice (n = 10) were injected i.p. with LPS (0.75 and 1.5 mg per mouse). (C) Acute septic peritonitis was induced by CLP. CLP and sham operations were performed as described in Methods on age-matched male Nrf2+/+ (n = 10) and _Nrf2_–/– mice (n = 10). Mortality was assessed every 12 hours for 5 days. *Nrf2+/+ mice showed improved survival compared wi Nrf2–/– mice. P < 0.05.
LPS elicits greater pulmonary inflammation in Nrf2-deficient mice. Because it is clear that Nrf2 is critical for survival during lethal septic shock, the role of this transcription factor in regulating nonlethal inflammatory stimulus was investigated. Lungs were examined after systemic (i.p. injection of 60 μg/mouse) or local (intratracheal instillation of 10 μg/mouse) administration of LPS. For both modes of LPS administration, the inflammatory response was greater in the lungs of _Nrf2_–/– mice than in their wild-type littermates. The influx of inflammatory cells (neutrophils and macrophages) was greater in the lungs of _Nrf2_–/– mice at both 6 and 24 hours after LPS challenge by either route. After i.p. administration of LPS, macrophages were the predominant cell type in bronchoalveolar lavage (BAL) fluid although both macrophages and neutrophils showed temporal increase in numbers (Figure 2, A and B). In contrast, intratracheal instillation attracted predominantly neutrophils (constituting as much as 80% of the total inflammatory cell population) in BAL fluid (Figure 2C), a finding similar to that of other investigators (13). Consistent with the BAL fluid analysis, histopathology showed a greater recruitment of inflammatory cells in perivascular, peribronchial, and alveolar spaces of _Nrf2_–/– mice 24 hours after LPS treatment (Figure 2D). Immunohistochemical examination of LPS-instilled lungs with anti-neutrophil antibody also confirmed a greater number of neutrophils in the lungs of _Nrf2_–/– mice (Figure 2E), which was further evident from myeloperoxidase activity in these lungs (Figure 2F). As a marker of lung injury, pulmonary edema was observed to be markedly higher in _Nrf2_–/– mice 24 hours after LPS instillation (Figure 2G). A similar pattern of lung pathological injury was induced by systemic delivery of LPS (data not shown). Taken together, these results show that disruption of the Nrf2 gene augments the innate immune response to bacterial endotoxin.
Nonlethal dose of LPS induced greater lung inflammation in Nrf2-deficient lungs. (A and B) BAL fluid analysis of _Nrf2_–/– and Nrf2+/+ mice after 6 and 24 hours of i.p. injection of LPS (60 μg per mouse). (C) BAL fluid analysis of _Nrf2_–/– and Nrf2+/+ mice after 6 hours and 24 hours of LPS instillation (10 μg per mouse). (D) Histopathological analysis of lungs by H&E staining 24 hours after instillation of LPS. Arrows indicate accumulation of inflammatory cells in the alveolar spaces. Magnification, ×20. (E) Immunohistology of lungs of both genotypes using anti-mouse neutrophil antibody 24 hours after LPS instillation. Sections were counterstained with hematoxylin. Arrows indicate neutrophils. Magnification, ×40. (F) Myeloperoxidase (MPO) activity in lung homogenates of both genotypes 6 and 24 hours after LPS instillation. (G) Pulmonary edema was assessed by the ratio of wet to dry lung weight 24 hours after LPS instillation. Data are presented as mean α SEM; n = 5. *Differs from vehicle control of the same genotype. †Differs from LPS-treated wild-type mic P < 0.05.
LPS and CLP induced greater secretion of TNF-α in Nrf2-deficient mice Since TNF-α is one of the early proinflammatory cytokines that is elevated during LPS- and CLP-induced inflammation, serum concentrations of TNF-α were measured by ELISA. After 1.5 hours of LPS challenge (1.5 mg/mouse), serum TNF-α was significantly higher in _Nrf2_–/– mice compared with Nrf2+/+ (Figure 3A). Similarly, after 6 hours of CLP, serum levels of TNF-α were greater in _Nrf2_–/– compared with Nrf2+/+ mice (Figure 3B). Furthermore, TNF-α concentrations in BAL fluid were also greater 2 hours after nonlethal LPS challenge (i.p. and intratracheal instillation) in _Nrf2_–/– mice compared with wild-type mice (Figure 3C). Next, we measured the concentrations of TNF receptors, TNFRI (p55) and TNFRII (p75), in Nrf2+/+ and _Nrf2_–/– mice after a lethal dose of LPS. While there was no difference in the constitutive serum levels of p55 and p75, after 6 hours of LPS treatment, the serum concentrations of both receptors were increased significantly. However there were no significant differences in the TNF receptors between the _Nrf2_–/– and _Nrf2+/+_mice (Supplemental Figure 1; supplemental material available online with this article; doi:10.1172/JCI25790DS1) after LPS challenge.
LPS and CLP induce greater secretion of TNF-α in Nrf2-deficient mice. (A) Serum concentration of TNF-α in Nrf2+/+ and _Nrf2_–/– mice 1.5 hours after LPS injection (1.5 mg per mouse). (B) Serum concentration of TNF-α in Nrf2+/+ and _Nrf2_–/– mice 6 hours after CLP. (C) TNF-α levels in BAL fluid at 2 hours after LPS delivery by i.p. injection (60 μg per mouse) and/or intratracheal instillation (10 μg per mouse). TNF-α in the BAL fluid of vehicle-treated mice was not detectable. Data are presented as mean α SEM. *Differs from vehicle control of the same genotype. †Differs from LPS-treated wild-type mice. P < 0.05. ND, n detected.
Temporal global changes in gene expression reflect the impact of Nrf2 on the innate immune response. Moderate increase in TNF-α production alone cannot explain the markedly higher CLP- and LPS- induced mortality as well as LPS-induced lung inflammation in _Nrf2_–/– mice (14). To systematically understand the role of Nrf2 during LPS-induced inflammation, the global gene expression profiles were examined in lungs of _Nrf2_–/– and Nrf2+/+ mice over time in response to a nonlethal LPS stimulus. After i.p. injection of LPS, microarray analyses of lungs were performed at 30 minutes, 1 hour, 6 hours, 12 hours, and 24 hours. Nrf2 deficiency resulted in the enhanced expression of several clusters of genes associated with the innate immune response, even at as early as 30 minutes (Figure 4). These included specific cytokines, chemokines, and cell surface adhesion molecules and receptors, among others. Differences between genotypes in expression of most of the proinflammatory genes in the lungs of mice were significant at the early time points (30 minutes and 1 hour) following LPS challenge. At later time points, with few exceptions, there was no significant difference in expression of proinflammatory genes between the genotypes. Henceforth, unless otherwise stated, a more detailed presentation of the gene expression profile obtained at 30 minutes is provided while the remaining data for the time course is presented as supplemental data. The microarray results indicate that Nrf2 functionality is indispensable for controlling the early surge of a large number of proinflammatory genes associated with the innate immune response.
Greater expression of proinflammatory genes associated with the innate immune response in the lungs of Nrf2-deficient mice. (A–C) Expression of cytokines (A), chemokines (B), and adhesion molecules/receptors (C) 30 minutes after nonlethal i.p. injection of LPS (60 μg per mouse) in _Nrf2_-deficient and wild-type mice. Gene expression data were obtained from microarray analysis. Data are represented as mean fold change obtained from comparing LPS-challenged to vehicle-treated lungs of the same genotype on a semilog scale. All the represented fold change values of LPS-treated lungs of _Nrf2_–/– mice are significant compared with wild-type mice at P <0.05.
Cytokines and chemokines. At 30 minutes after LPS challenge, gene expression for cytokines such as TNF-α, TNFSF9, IL-1_α, IL-6, IL1F9, IL-10, IL-12_β_, IL-23p19, CSF1,_ and CSF2 was significantly higher in lungs of _Nrf2_–/– compared with Nrf2+/+ mice. Among all cytokines, the expression of IL-6 was highest. Members of C-C family (CCL12 [_MCP5_], CCL17 [_TARC_], CCL2 [_MCP1_], CCL3 [_MIP1_α], CCL4 [_MIP1_β], and CCL6 and CCL8 [_MCP2_]) and C-X-C chemokines (MIP2, MIG, KC, ITAC, IP-10, and CXCL13) were greatly upregulated in LPS-challenged _Nrf2_–/– lungs relative to Nrf2+/+ (Figure 4 and Supplemental Table 1).
Cell surface adhesion molecules and receptors. Disruption of Nrf2 had no effect on the expression of the LPS signaling receptor, TLR4, after LPS challenge. CD14 transcript was markedly higher in _Nrf2_–/– lungs. Expression of several adhesion molecules, such as PGLYRP1 (a member of the peptidoglycan recognition protein family; ref. 15), TREM-1, SELE, SELP, VCAM1, and members of the C-type lectin family (CLEC4D, CLEC4E) were highly upregulated in _Nrf2_–/– lungs (Supplemental Table 2). C5AR, which mediates C5A response and augments sepsis (16), was upregulated to a greater extent in _Nrf2_–/– mice (Supplemental Table 2). Among the cell surface adhesion molecules, TREM1 and CD14 were highly upregulated in _Nrf2_–/– lungs.
Regulators of cytokine signaling and transcription. Transcripts of SOCS3, which are involved in downregulating cytokine signaling, were induced to a greater extent in _Nrf2_–/– lungs at early time points (Supplemental Table 1). Transcription factors belonging to the NF-κB family (C-RELC, RELB, NFKBIZ, NFKB2, NFKBIE), the interferon family (IRF5, IRF1, IFI202B, IFI204, IRF1), and the early growth response family (EGR2, EGR3) as well as STAT4, which collectively regulate different inflammatory cascade pathways, were expressed to higher levels in _Nrf2_–/– lungs when compared with wild-type mice (Supplemental Table 3).
Immunoglobulin and MHC. Transcripts of many members of the immunoglobulin family (IGHG, IGH-VJ558, IGH-4, IGH-6, IGJ, IGK-V21, IGk-V32, IGK-V8, IGL-V1, IGSF6, and IGM) as well as the MHC class II family (H2-AA, H2-AB1, H2-EA, H2-DMA, H2-DMB1, and H2-DMB2) were selectively upregulated in the lungs of Nrf2–/– mice at 30 minutes (Supplemental Table 4), indicating severe immune dysfunction.
Acute phase proteins, heat shock proteins, and other inflammation-modulating molecules and enzymes. Many genes that encode for acute phase proteins such as proteinase inhibitors (SERPINA3M, SERPINB2, and SERPINE1), serum amyloid A (SAA2, SAA3), orsomucoid (ORM1, ORM2), and HSP1A were markedly increased in _Nrf2_–/– lungs (Supplemental Table 5). Expression levels of ARG2 (an endogenous inhibitor of iNOS that regulates arginine metabolism; ref. 17), INDO (exerts immunosuppressive effects through induction of apoptosis in T cells by regulating tryptophan metabolism; ref. 18), PLEK (regulates phagocytosis activity by macrophages; ref.19), and PFC (regulator of alternative complement system) were all higher in Nrf2–/– lungs at 30 minutes (Supplemental Table 6).
ROS/RNS generators. The expression of NCF1 (p47phox) and NCF4 (p40phox), which are members of the NADPH oxidase family involved in generation of reactive oxygen species during phagocytic activity by neutrophils and macrophages, were significantly higher in _Nrf2_–**/–** lungs at early stages (until 1 hour; Supplemental Table 6). Expression of NOS2 (iNOS), which is involved in nitric oxide generation, was induced at the 6-hour time point and was greater in the lungs of _Nrf2_–**/–** mice (Supplemental Table 6).
Antioxidants. Nrf2 is a key transcription factor for regulating the expression of antioxidative genes. Differential gene expression profiling of vehicle-treated Nrf2+/+ and _Nrf2_–/– lungs showed constitutively elevated expression of antioxidative genes such as glutathione peroxidase 2 (GPX2), glutamate cysteine ligase catalytic subunit (GCLC), thioredoxin reductase 1, and members of the glutathione _S_-transferase family in wild-type mice (Supplemental Table 7). Although expression of these genes was not altered significantly in wild-type mice after LPS challenge, at all time points, transcript levels of these antioxidative genes were higher in the lungs of wild-type mice compared with _Nrf2_–/– mice.
Validation of microarray data by real-time quantitative PCR. Genes that were selected for validation included chemokines (MCP5, MCP1, and MIP2), cytokines (IL-6, _IL-1_α, _TNF-_α, and CSF2), an LPS membrane receptor (CD14), immunoglobulins (IGH-4 and IHSF6), an MHC class II member (H2-EA), and the transcription factor STAT4. Expression values of these genes obtained from real-time PCR were consistent with the microarray values in terms of magnitude and pattern across all time points (Supplemental Table 8).
_TNF-_α stimulus induces a greater pulmonary inflammatory response in Nrf2-deficient mice. Microarray and BAL fluid analysis showed greater expression of TNF-α in the lungs of _Nrf2_–/– mice compared with Nrf2+/+ mice in response to LPS. To characterize the effect of TNF-α–mediated inflammation, mice of both genotypes were administered TNF-α (i.p.). Following TNF-α treatment, lungs of _Nrf2–/–_mice showed increased infiltration of inflammatory cells as measured by BAL fluid analysis and histopathology (Figure 5, A and B) when compared with wild-type littermates. Real-time PCR analysis of selected genes (_TNF-_α, _IL-1_β, and IL-6) in the lungs of mice 30 minutes after TNF-α treatment revealed greater expression in _Nrf2_–/– mice compared with Nrf2+/+ (Figure 5C). Taken together, as with LPS, treatment with TNF-α induced greater inflammation in _Nrf2_–/– lungs.
TNF-α stimulus induced greater lung inflammation in Nrf2-deficient mice. (A) BAL fluid analysis at 6 hours after i.p. injection of TNF-α (10 μg/mouse). (B) Histopathological analysis of lungs of Nrf2+/+ and _Nrf2_–/– mice by H&E staining 24 hours after i.p. injection of TNF-α (10 μg/mouse). Vehicle-treated lungs are not shown. Magnification, ×20. (C) Gene expression analysis of TNF-α, IL-1β, and IL-6 by real-time PCR in the lungs of _Nrf2_–/– and Nrf2+/+ mice 30 minutes after TNF-α challenge. Data are presented as mean α SEM. *Differs from vehicle control of the same genotype. †Differs from LPS-treated wild-type mice. < 0.05.
NF-κB activity is greater in lungs of LPS-treated Nrf2-deficient mice. Because the lungs of _Nrf2_–/– mice showed greater infiltration of inflammatory cells and higher expression of largely inflammation-associated genes, we assessed NF-κB activity, which regulates the expression of several genes that are essential for initiating and promoting inflammation (20). At 30 minutes after LPS instillation, NF-κB DNA-binding activity was significantly higher in nuclear extracts from lungs of _Nrf2_–/– mice than in their wild-type counterparts, suggesting an inhibitory role of Nrf2 on NF-κB activation (Figure 6, A and B). Western blot analysis confirmed a greater increase in nuclear levels of p65, an NF-κB subunit, in the LPS-treated lungs of _Nrf2_–/– mice than in _Nrf2+/+_mice (Figure 6, C and D). Similarly, nuclear extracts from the lungs of _Nrf2_–/– mice showed increased binding of p65/v-rel reticuloendotheliosis viral oncogene homolog A (p65/RelA) subunits to NF-κB–binding sequence as measured by ELISA using Mercury TransFactor ELISA kit (Supplemental Figure 3A). A similar trend toward increased NF-κB activation in _Nrf2_–/– mice was observed at 30 minutes and 1 hour following i.p. injection of LPS at a nonlethal dose (data not shown).
LPS induced greater NF-κB activation in lungs of Nrf2-deficient mice. (A) Lung nuclear extracts from _Nrf2_–/– and Nrf2+/+ mice were assayed for NF-κB DNA-binding by EMSA 30 minutes after instillation of LPS (10 μg per mouse). The major NF-κB bands contained p65 and p55 subunits, as determined by the supershift obtained by p65 and p50 antibody. Lanes: 1, vehicle, Nrf2+/+; 2, LPS, Nrf2+/+; 3, vehicle, _Nrf2_–/–; 4, LPS, _Nrf2_–/–; 5, LPS, Nrf2+/+ with p65 antibody, 6, LPS, Nrf2+/+ with p50 antibody. SS, supershift. (B) Quantification of NF-κB DNA-binding was performed by densitometric analysis. All values are mean α SEM obtained from 3 animals per treatment group and are represented as relative to respective vehicle control. (C) Nuclear accumulation of p65 by Western blot in the nuclear extracts derived from lungs of Nrf2+/+ and _Nrf2_–/– mice 30 minutes after instillation of LPS (10 μg/mouse). Lamin B1 was used as loading control. (D) Densitometric analysis of Western blot of RelA relative to wild-type vehicle control. All values are mean α SEM; n = 3. *Differs from vehicle control of the same genotype. †Differs from LPS-treat wild-type mice. P < 0.05.
LPS induces greater NF-κB activity and TNF-α secretion in peritoneal macrophages from Nrf2-deficient mice Macrophages play a central role in immune dysfunction during endotoxic shock. To examine the effect of Nrf2 deficiency on NF-κB activation in macrophages, resident peritoneal macrophages were stimulated with LPS. After 20 minutes, the DNA-binding activity of NF-κB was substantially higher in _Nrf2_–/– macrophages than in the wild-type counterparts as determined by EMSA (Figure 7, A and B). The greater increase in NF-κB activity in _Nrf2–/–_macrophages correlated well with the increase in TNF-α levels measured 0.5 hours, 1 hour, and 3 hours after LPS treatment (Figure 7C).
Lack of Nrf2 augments NF-κB activation in macrophages. (A) Nuclear extracts of Nrf2+/+ and _Nrf2_–/– peritoneal macrophages were assayed for NF-κB DNA-binding by EMSA 20 minutes after LPS treatment (1 ng/ml). (B) Densitometric analysis of NF-κB DNA-binding relative to wild-type vehicle control. Values are mean α SEM; n = 3. (C) TNF-α levels in the culture media from _Nrf2+/+_and _Nrf2–/–_peritoneal macrophages after 0.5 hours, 1 hours, and 3 hours of LPS treatment (1 ng/ml). *Differs from vehicle control of the same genotype. †Differs from wild-type treatment grou P < 0.05.
Increased NF-κB activation by LPS or TNF-α in Nrf2-deficient mouse embryonic fibroblasts To further probe the role of Nrf2 in regulating NF-κB, mouse embryonic fibroblasts (MEFs) derived from _Nrf2_–/– and Nrf2+/+ mice were exposed to LPS or TNF-α. Both LPS and TNF-α stimulation resulted in enhanced activation of NF-κB in _Nrf2_–/– MEFs compared with Nrf2+/+ cells as measured by EMSA (Figure 8A). There were 3- and 5-fold increases in NF-κB activation in _Nrf2_–/– MEFs relative to wild-type in response to LPS or TNF-α stimulation, respectively (Figure 8B). The specificity of NF-κB binding was assessed by adding an excess of cold mutant NF-κB oligo to the binding reactions. Supershift analysis of nuclear extracts from LPS- and TNF-α–treated _Nrf2–/–_MEFs with p65 and p50 antibodies demonstrated heterodimers of p50 and p65. Nuclear extracts from the _Nrf2_–/– MEF cells treated with LPS or TNF-α also demonstrated increased binding of p65/RelA subunits to NF-κB binding sequence as determined by an ELISA-based method of detecting NF-κB DNA-binding activity using Mercury TransFactor ELISA kit (Supplemental Figure 3B). NF-κB–mediated luciferase reporter activity was also greater in _Nrf2_–/– MEFs than in _Nrf2+/+_MEFs in response to LPS or TNF-α (Figure 8C). In general, the _Nrf2_–/– MEFs showed greater NF-κB activation in response to TNF-α compared with LPS stimulation.
LPS and/or TNF-α stimulus induces greater NF-κB activation in Nrf2-deficient MEFs. (A) Nuclear extracts from Nrf2+/+ and _Nrf2_–/– MEFs were assayed for NF-κB DNA-binding activity by EMSA 30 minutes after LPS (0.5 μg/ml) and or TNF-α (10 ng/ml). The major NF-κB bands contained p65 and p55 subunits, as determined by the supershift analysis using p65 and p55 antibody. (B) Quantification of NF-κB DNA-binding was performed by densitometric analysis. All values are mean α SEM (n = 3) and are represented relative to respective vehicle control. (C) NF-κB–mediated reporter activity in MEFs of both genotypes challenged with LPS (0.5 μg/ml) and TNF-α (10 ng/ml). At 24 hours after transfection with p–NF-κB–Luc vector, cells were treated with LPS and/or TNF-α for 3 hours, and then luciferase activity was measured. Data are mean α SEM from 3 independent experiments (n = 3) and are represented relative to respective vehicle control. (D) Immunoblot of IκB-α and p–IκB-α protein in Nrf2+/+ and _Nrf2_–/– MEFs after LPS (0.5 μg/ml) or TNF-α (10 ng/ml) stimulus. (E and F) Quantification of IκB-α (E) and p–IκB-α (F) protein in Nrf2+/+ and _Nrf2_–/– MEFs by densitometric analysis. Data are mean α SEM (n = 3) and are shown as relative to respective vehicle control. (G) IKK activity in Nrf2+/+ and _Nrf2_–/– MEFs after LPS (0.5 μg/ml) or TNF-α (10 ng/ml) stimulus. (H) Quantification of IKK activity in Nrf2+/+ and _Nrf2_–/– MEFs by densitometric analysis. Densitometric units are normalized to IKKα. Data are mean α SEM (n = 3) and are relative to respective controls. *Differs from vehicle control of the same genotype. †Differs frowild-type treatment group. P < 0.05.
Nrf2 regulates NF-κB activation by modulating IκB-α degradation. To understand the mechanism of augmented NF-κB activation in _Nrf2_–/– MEFs, IκB-α and phosphorylated IκB-α (p–IκB-α) were measured in the whole cell extracts of _Nrf2_–/– and Nrf2+/+ MEFs after treatment with LPS or TNF-α. In response to LPS or TNF-α, IκB-α degradation was significantly higher in _Nrf2_–/– MEFs compared with wild-type cells (Figure 8, D and E). TNF-α stimulus induced greater phosphorylation of IκB-α while LPS induced moderate but statistically significant increases in phosphorylation of IκB-α in _Nrf2_–/– MEFs compared with Nrf2+/+ MEFs (Figure 8, D and F). Furthermore, activity of IκB kinase (IKK) kinase, which regulates phosphorylation of IκB-α, was also greater in _Nrf2_–/– MEFs in response to LPS or TNF-α (Figure 8, G and H).
Nrf2 affects both MyD88-dependent and MyD88-independent signaling. Microarray gene expression analysis after LPS challenge revealed that, in addition to NF-κB–regulated genes, several interferon regulatory factor 3–regulated (IRF3-regulated) genes (such as IP-10, MIG, ITAC, and ISG54; Supplemental Table 9) were expressed to a greater magnitude in the lungs of _Nrf2_–/– mice. LPS via TLR4 can activate MyD88-dependent signaling leading to NF-κB activation as well as MyD88-independent signaling (TRIF/IRF3), resulting in IRF3 activation (21). As shown in Figure 8C, Nrf2 deficiency greatly upregulates NF-κB–mediated luciferase activity in MEFs in response to LPS, suggesting the effect on MyD88-dependent signaling. In order to understand the influence of Nrf2 deficiency on MyD88-independent signaling, MEFs of both genotypes were transfected with a luciferase reporter vector containing interferon-stimulated response element (ISRE) and treated with LPS or polyinosinic-polycytidylic acid [poly(I:C)]. LPS elicited greater IRF3-mediated luciferase reporter activity in _Nrf2_–/– MEFs compared with Nrf2+/+ MEFs (Figure 9). Similarly, in response to poly(I:C), which acts specifically via MyD88-independent signaling (22), IRF3-mediated reporter activity was significantly higher in _Nrf2_–/– MEFs (Figure 9).
Nrf2 deficiency increases LPS- and or poly(I:C)-induced IRF3-mediated luciferase reporter activity in MEFs. At 24 hours after transfection with ISRE-Tk-Luc vector, cells were treated with LPS and or poly(I:C) for 6 hours, and luciferase assays were performed 6 hours after treatment. For poly(I:C) stimulation, MEFs were transfected with 6 μg of poly(I:C) in 8 μl of Lipofectamine2000. Data are mean α SEM from 3 independent experiments; n = 3. *Differs from vehicle control of the same genotype; †Differs from wild-type treatment group. P <0.05.
Glutathione levels are lower in lungs and MEFs of Nrf2-deficient mice. Nrf2 is a regulator of a battery of cellular antioxidants, including the glutathione-synthesizing (GSH-synthesizing) enzyme, glutamate cysteine ligase. Constitutive expression of GCLC was significantly lower in the lungs as well as MEFs of _Nrf2_–/– mice compared with Nrf2+/+ mice (Figure 10A). This difference in expression is reflected in significantly lower endogenous levels of GSH in the MEFs of _Nrf2–/–_mice than in Nrf2+/+ mice (Figure 10D). In response to LPS stimulus, there was a significant decrease in the levels of GSH in MEFs of both genotypes at 1 hour (Figure 10D). In contrast, after 24 hours of LPS treatment, a greater increase in GSH was observed in the lungs of Nrf2+/+ mice compared with Nrf2–/–(Figure 10B). The ratio of GSH to oxidized GSH (GSSG) after LPS challenge was significantly higher in the lungs of wild-type mice, implying greater amounts of GSSG in _Nrf2_–/– lungs and thus a difference in redox status between the 2 genotypes (Figure 10C).
Lower levels of GSH in the lungs and MEFs of Nrf2-deficient mice. (A) Constitutive expression of GCLC in lungs and MEFs of Nrf2+/+ and _Nrf2_–/– mice. (B) GSH levels in the lungs of mice of both genotypes 24 hours after LPS instillation (10 μg per mouse). Data are mean α SEM from 3 independent experiments and are expressed as percentage increases relative to vehicle-treated Nrf2+/+ group. (C) Ratio of GSH to GSSG measured 24 hours after LPS instillation in the lung of Nrf2+/+ and _Nrf2_–/– mice. Data are mean α SEM from 3 independent experiments. (D) GSH levels in Nrf2+/+ and _Nrf2_–/– MEFs at 1 hour after LPS (0.5 μg/ml) or TNF-α (10 ng/ml) stimulus. Data are presented as mean α SEM; n = 4. *Differs from vehicle control of the same genotype. †Differs from wild-type treatmt group. P < 0.05.
N-acetyl cysteine and GSH-monoethyl ester decrease LPS- and TNF-α–induced NF-κB activation in Nrf2-deficient MEFs. To investigate whether replenishing antioxidants could suppress the enhanced NF-κB activation observed in _Nrf2_–/– cells, MEFs transfected with NF-κB–Luc reporter vector were pretreated with N_-_acetyl cysteine (NAC) or GSH-monoethyl ester for 1 hour and then challenged with LPS or TNF-α. Pretreatment with NAC or GSH-monoethyl ester significantly attenuated NF-κB–mediated reporter activity in _Nrf2_–/– cells elicited in response to LPS or TNF-α (Figure 11A).
Pretreatment with exogenous antioxidants alleviates inflammation in Nrf2-deficient mice. (A) NF-κB–mediated luciferase reporter activity in _Nrf2_–/– MEFs pretreated for 1 hour with NAC (10 mM) and/or GSH-monoethyl ester (GSH) (1 mM) after 3 hours of LPS (0.5 μg/ml) and/or TNF-α (10 ng/ml) stimulus. Data are presented as mean α SEM (n = 4). *Differs from vehicle control. †Differs from group that was treated with LPS or TNF-α only. P < 0.05. (B) Expression of TNF-α, IL-1β, and IL-6 by real-time PCR at 30 minutes in the lungs of _Nrf2_–/– mice pretreated with NAC after LPS (i.p., 60 μg per mouse) challenge. (C) BAL fluid analysis at 6 hours in lungs of _Nrf2_–/– mice pretreated with NAC after LPS (i.p., 60 μg per mouse) challenge. _Nrf2_–/– mice were pretreated with 3 doses of NAC (500 mg/kg body weight, i.p., every 4 hours). Data are presented as mean α SEM. n = 4. #Differs from only LPS treatment. P < 0.05. (D) LPS-induced mortality in _Nrf2_–/– and _Nrf2+/+_mice pretreated with NAC. Age-matched male Nrf2–/–(n = 10) and Nrf2+/+ mice (n = 10) were pretreated with NAC (i.p., 500 mg/kg body weight) and/or saline every day for 4 days followed by LPS challenge (1.5 mg per mouse). Mortality was assessed every 12 hours for 5 days. **Mice pretreated with NAC had improved survival comped with vehicle-pretreated mice. P < 0.05.
NAC abrogates LPS-induced proinflammatory gene expression in the lungs of Nrf2-deficient mice and protects against lethality. Since LPS challenge enhanced the expression of several NF-κB–regulated proinflammatory genes in lungs of _Nrf2_–/– mice compared with wild-type littermates, we investigated whether administration of an exogenous antioxidant could attenuate this augmented proinflammatory cascade. Mice were pretreated with NAC (500 mg/kg body weight) and then challenged with nonlethal doses of LPS. After 30 minutes of LPS challenge, selected proinflammatory genes were measured by real-time PCR analysis. Transcript levels of _TNF-_α, _IL-1_β, and IL-6 were significantly reduced in the lungs of _Nrf2–/–_mice by pretreatment with NAC (Figure 11B). Concordantly, the influx of inflammatory cells was also significantly reduced by pretreatment of _Nrf2_–/– mice with NAC (Figure 11C). We next investigated to determine whether exogenous NAC supplementation could protect against LPS-induced septic shock in _Nrf_2–/– mice. Mice of both genotypes were pretreated with NAC (500 mg/kg body weight) for 4 days prior to LPS challenge (1.5 mg per mouse). All _Nrf2_–/– mice pretreated with saline died within 56 hours while 40% of mice pretreated with NAC survived (Figure 11D). Pretreatment of wild-type mice with NAC provided modest protection. These results suggest that exogenous antioxidants such as NAC can partially ameliorate the phenotype of _Nrf2–/–_mice.