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Monocytes play a central role in the immunopathological effects of sepsis. This role is mediated by production of the cytokines tumor necrosis factor α (TNF) and interleukin-1β (IL-1β). The transcription factor NF-E2-related factor 2 (Nrf2) regulates innate immune responses in various experimental disease models. Presently, the role of Nrf2-regulated genes in lipopolysaccharide (LPS)-treated human monocytes is not well defined. Here we show that Nrf2 mediates a significant regulation of LPS-induced inflammatory responses. Analysis of Nrf2-regulated gene expression in human monocytes showed that LPS induced the expression of the phase II detoxification gene NAD(P)H:quinone oxidoreductase 1 (NQO1). Furthermore, NQO1 mRNA or protein expression in response to LPS was regulated by Nrf2. Silencing Nrf2 expression in human monocytes inhibited LPS-induced NQO1 expression, however by contrast, it significantly increased TNF and IL-1β production. Silencing expression of NQO1 alone, or in combination with heme oxygenase-1 (HO-1) silencing, markedly increased LPS-induced TNF and IL-1β expression. Additionally, overexpression of NQO1 and/or HO-1 inhibited LPS-induced TNF and IL-1β expression. These results show for the first time that LPS induces NQO1 and HO-1 expression in human monocytes via Nrf2 to modulate their inflammatory responsiveness, thus providing novel potential therapeutic strategies for the treatment of sepsis.
Sepsis is a major public health problem throughout the world. It affects 30-50% of intensive care unit patients (1-3), with an annual incidence of approximately 250 cases per 100,000 population in the Western world (4). Sepsis is characterized by a systemic inflammatory response caused by bacterial infection or trauma. The most common bacterial component implicated in initiating the septic syndrome is a cell wall molecule derived from gram-negative bacteria, known as lipopolysaccharide (LPS)(5). The inflammatory responses induced by LPS are predominantly controlled by monocytes and macrophages which produce the prototypic inflammatory cytokines tumor necrosis factor α (TNF) and interleukin 1β (IL-1β), and these in turn mediate many of the immunopathological features of sepsis (6,7).
Host factors that regulate inflammatory responses may protect against dysfunctional inflammation during bacterial infection. The basic leucine zipper transcription factor, NF-E2-related factor 2 (Nrf2), which when activated, induces the expression of a host of cytoprotective and detoxification genes, is a known regulator of inflammatory responses (8-10). In endothelial cells, Nrf2 activation inhibits pro-inflammatory cytokine-induced adhesion molecule expression (11). In a mouse model of experimental sepsis, exposure of Nrf2-deficient mice to endotoxin leads to increased TNF and interleukin-6 expression when compared to wildtype animals (12). In addition, the synthetic triterpenoid CDDO-Im inhibits LPS-induced inflammation by inducing Nrf2-regulated genes in mice (13). Moreover, activation of Nrf2 protects inflammatory cells from oxidant injury and inhibits pro-inflammatory gene expression (14). Taken together, these studies suggest that Nrf2 plays an important role in modulating the magnitude of inflammatory responses. However, the mechanism by which Nrf2 mediates this protection remains to be elucidated.
Monocytes play a key role in mobilization of the immune response during sepsis (15). In response to LPS, monocytes produce both pro-inflammatory mediators and regulatory proteins that counteract the inflammation and oxidative stress. Indeed, we have demonstrated that LPS activation of human monocytes induces the expression of the cytoprotective protein heme oxygenase-1 (HO-1)(16). HO-1 induction was shown to be induced by Nrf2 activation. In addition, others have shown that exposure of HO-1-deficient mice to endotoxin leads to increased splenic pro-inflammatory cytokine secretion, and higher mortality from endotoxic shock when compared with wildtype animals (17,18). Taken together, these studies suggest that HO-1 and its transcription factor Nrf2 play an important role in counteracting deleterious increases in inflammation and oxidative injury.
Nrf2 is ubiquitously expressed in animal tissues and increasing evidence reveals that Nrf2 mediates antioxidant response element (ARE)-dependent gene transcription (8). The genes regulated by Nrf2 include phase II enzymes such as NAD(P)H:quinone oxidoreductase 1 (NQO1), glutathione S-transferase (GST), and glutathione reductase (GR) which detoxify endogenous and exogenous chemicals through reduction and conjugation reactions (19). It also includes the regulation of glutamyl cysteine ligase modulatory (GCLM) and catalytic (GCLC) units, the two sub-units of the rate-limiting enzyme in glutathione biosynthesis. Several of these Nrf2-regulated genes have been shown to have anti-inflammatory actions (8,14,20,21), which suggests that Nrf2 mediates its control on inflammation by inducing the expression of any number of ARE-dependent genes.
The molecular mechanism of Nrf2 action is yet to be fully resolved. There are two reported mechanisms proposed regarding Nrf2 activation. Firstly Nrf2 is bound to its repressor Keap1 which inhibits its activity and upon activation, Nrf2 is released from Keap1 and translocates into the nucleus (22,23). The second mechanism is that under normal basal conditions Keap1 regulates the ubiquitin-26S proteasome-mediated turnover of Nrf2. Upon activation Keap1 stabilizes Nrf2 which translocates into the nucleus (24,25). Once in the nucleus Nrf2 forms a heterodimer with Maf proteins (26). The heterodimer then binds to ARE located in the enhancer regions and mediates the transcription of the Nrf2-inducible genes (27).
In the present study, we report that exposure of human monocytes to LPS activates Nrf2 transcriptional activity and increases the expression of NQO1. We further demonstrate that transcriptional activity of Nrf2 is mediated by Nrf2 mRNA expression induced by LPS. Previously we have shown that LPS can induce the expression of HO-1 in human monocytes (16). Here we show that silencing the expression of NQO1 and HO-1 prolongs LPS-induced TNF and IL-1β expression. These data suggest that LPS exerts protective effects through the co-ordinated expression of Nrf2-regulated genes in human monocytes. These findings provide an important new insight into the regulation of inflammation in human monocytes.
The human monocytic cell line THP-1 was obtained from the European Collection of Cell Cultures. Human HO-1 antibody was purchased from StressGen Biotechnologies. All other antibodies were obtained from Santa Cruz Biotechnology. LPS was procured from Calbiochem. Nrf2, NQO1 and HO-1 siRNA were purchased from Ambion. All other reagents were obtained from Sigma–Aldrich.
Primary human monocytes and THP-1 cells were cultured in RPMI 1640 medium supplemented with 10% FCS, 2 mM L-glutamine (Invitrogen), and 2-ME. Cells were maintained in a humidified atmosphere at 37°C and 5% CO2. For monocyte isolation, heparinized blood was collected from healthy volunteers and human PBMCs isolated by Percoll (Amersham Pharmacia Biotech) density gradient centrifugation (28). PBMCs (4 × 106/ml) were incubated in complete medium for 2 h at 37°C to allow adherence of monocytes (29). Cell type was confirmed by microscopy and flow cytometry.
To construct the HO-1 mammalian expression vectors, full-length human HO-1 was generated by RT-PCR and cloned into pcDNA3 to generate pcDNA3HO-1. pcDNA3NQO1 was kindly provided by X-L Chen (Atherogenics, USA)(11).
Total RNA was extracted from 5 × 105 treated THP-1 or primary monocytic cells using the Nucleic Acid PrepStation from Applied Biosystems, according to the manufacturer’s instructions. Reverse transcription was performed using the RNA PCR core kit (Applied Biosystems). Real-time PCR primers for GAPDH, HO-1, IL-1β, NQO1, GCLM, GSTA1, GR, Nrf2, TNF, thioredoxin reductase 1 (TrxR1), GCLC GSTA2 and interleukin-10 (IL-10) were purchased from Invitrogen. Sequences of primers were as follows: GAPDH forward, 5′-ACCAGCCTCAAGATCATCAGCA-3′; GAPDH reverse, 5′-TGCTAAGCAGTTGGTGGTGC-3′; HO-1 forward, 5′-ATGGCCTCCCTGTACCACATC-3′; HO-1 reverse, 5′-TGTTGCGCTCAATCTCCTCCT-3′; IL-1β forward 5′-CTGGACCTCTGCCCTCTGG-3′; IL-1β reverse 5′-TCCATGGCCACAACAACTGA-3′; ; NQO1 forward, 5′-CGCAGACCTTGTGATATTCCAG-3′; NQO1 reverse, 5′-CGTTTCTTCCATCCTTCCAGG-3′; GCLM forward, 5′-TGCAGTTGACATGGCCTGTT-3′; GCLM reverse, 5′-TCACAGAATCCAGCTGTGCAA-3′; GSTA1 forward, 5′-AGCCCAAGCTCCACTACTTCAAT-3′; GSTA1 reverse, 5′-CTTCAAACTCTACTCCAGCTGCAG-3′; GR forward, 5′-TTGTCGGGCTTGGAAGTCAG-3′; GR reverse, 5′-TGGTAGCCTACCGGGAACTG-3′; Nrf2 forward, 5′-AACCACCCTGAAAGCACAGC-3′; Nrf2 reverse, 5′-TGAAATGCCGGAGTCAGAATC-3′; TNF forward, 5′-GCCCAGGCAGTCAGATCATC-3′; TNF reverse, 5′-CGGTTCAGCCACTGGAGCT-3′; TrxR1 forward, 5′-GGGCAATTTATTGGTCCTCA-3; TrxR1 reverse, 5′-GGTCTTTCACCAGTGGCAAT-3; GCLC forward, 5′-GGATTTGGAAATGGGCAATTG-3′; GCLC reverse, 5′-CTCAGATATACTGCAGGCTTGGAA-3′; GSTA2 forward, 5′-ATGTTCCAGCAAGTGCCAATG-3′; GSTA2 reverse, 5′-TGAGAATGGCTCTGGTCTGCA-3′; IL-10 forward, 5′-TTACCTGGAGGAGGTGATGC-3′ IL-10 reverse, 5′-GGCCTTGCTCTTGTTTTCAC-3′. Relative quantitative real-time PCR used SYBR green technology (Sigma) on cDNA generated from the reverse transcription of purified RNA. After pre-amplification (95 °C for 2 min), the PCRs were amplified for 40 cycles (95°C for 15s and 60°C for 1 min) on a IQ5 Real-time PCR Detection System (Bio-Rad). Each mRNA expression was normalized against GAPDH mRNA expression using the comparative cycle threshold method.
TNFα protein expression was measured using the human TNFα ELISA set (BD Biosciences). SDS-PAGE and Western analyses were performed as described previously (29, 30). Nuclear extracts were prepared as described previously (31). Protein was transferred to nitrocellulose membrane and Western analyses were performed with the indicated antibody according to their manufacturer’s guidelines, using ECL detection. In some instances, immunoblot band intensity was measured by densitometric analysis using Quantity One software (BioRad).
NF-κB DNA-binding was measured using the NF-κB p65 Transcription Factor ELISA kit (Panomics). Nrf2 binding was measured by electrophoretic mobility shift assay. An oligonucleotide probe containing the human NQO1 ARE site (underlined), 5′ –TTCCAATCCGCAGTCACAGTGACTCAGCAGA-3′ was manufactured and biotinylated by Sigma-Genosys. For competition binding, the same sequence was manufactured without the biotin label (Sigma-Genosys). Nuclear extracts were prepared as previously described (16) and incubated with the biotin-labeled probes using the LightShift Chemiluminescent EMSA kit (Pierce), following the manufacturer’s instructions. For supershift analysis, nuclear extracts from treated THP-1 cells were pre-incubated with 1 μg of either anti-human Nrf2 or anti-human p65 supershift antibodies (Santa Cruz Biotechnology) for 20 min before gel shift analysis.
A dichlorofluorescein (DCF) assay was used to determine cellular ROS generation in THP-1 cells (32). Briefly, 106 cells were unstimulated or treated with 10μg/ml LPS for various times. Following this, cells were washed in PBS and then incubated with 10 μM 2′,7′-dichlorodihydrofluorescein diacetate (H2-DCFDA) (Molecular Probes) for 15 mins at 37°C and 5% CO2. Cells were collected and washed with RPMI containing 10mM HEPES and measured for oxidation of H2-DCFDA using a flow cytometer (Beckman Coulter Epics XL). The fluorescence intensity measuring the oxidation of H2-DCFDA by ROS represents the relative steady state of ROS generation in cells. Thus H2-DCFDA oxidation was calculated by dividing the mean channel fluorescence of a treated sample by that of the untreated one and multiplying by 100 to obtain the relative change, expressed as a percentage (33).
Primary monocytes and THP-1 cells (1 × 106/well) were transfected using Amaxa Nucleofector Technology. Equivalent molar concentrations of the siRNA were used to yield a final concentration of 30 nM. Transfected cells were incubated for 24 h. Following this, cells were stimulated as described above. THP-1 were transfected with various plasmids as indicated in the figure legends using 1μg of DNA for 1×106 cells using Amaxa Nucleofector Technology.
Student t test was performed to assess statistical significance from controls. Results with p<0.05 were considered statistically significant. Results are displayed as means ± SD of at least 3 independent experiments. For Western blotting and EMSA experiments, data are representative of 3 independent experiments.
Nrf2 is a key transcription factor in the regulation of antioxidant and cytoprotective genes (8). We have recently shown that HO-1 is regulated by Nrf2 in response to LPS (16). To determine if other Nrf2 regulated genes are induced by LPS in human monocytes we examined the mRNA expression of GCLM, GCLC, TrxR1, HO-1, NQO1, GR, GSTA1 and GSTA2 in response to LPS stimulation at 8 h and 24 h. LPS stimulated a significant increase in NQO1 mRNA expression as well as the positive control HO-1 in primary human monocytes (Figure 1A). No significant induction of GSTA1, GSTA2, GCLC, GCLM, GR and TrxR1 was seen (Figure 1A). Further analysis revealed that LPS induced NQO1 expression in the human monocytic cell line THP-1 and that LPS induced NQO1 mRNA expression by 4 h, increased further by 8 h, decreased by 12 h and further decreased by 16 and 24 h (Figure 1B). To confirm the mRNA results, NQO1 protein expression was examined by Western blot analysis. LPS-induced NQO1 protein increased in both THP-1 cells and human monocytes (Figure 1C). Immunoblots were reprobed with a mouse anti-human β-actin antibody to confirm equal loading between samples. These results demonstrate similar results in both primary monocytes and THP-1 cells, suggesting that the THP-1 cells are a valid model for studying LPS-induced Nrf2 activation in human monocytes.
How Nrf2 is activated in response to LPS is not clearly understood. As outlined above, the potential molecular mechanisms of Nrf2 activation could be either release of Nrf2 protein from its bound repressor Keap1, or the inhibition of Keap1-regulated ubiquitin-proteasome degradation of Nrf2, with either situation leading to Nrf2 translocation to the nucleus (24,25). To address this question, we first examined the expression of Nrf2 in response to LPS in whole cell extracts to determine if there are any changes in Nrf2 protein expression in response to LPS. Figure 2A shows that LPS induced the expression of Nrf2 protein in human THP-1 cells which peaks at 4 h, but was still above control levels at 24 h. These results suggested that Nrf2 protein is synthesized de novo. To confirm these results we examined the accumulation of Nrf2 in response to LPS in THP-1 cells after treatment with the protein synthesis inhibitor cycloheximide (CHX). While accumulation of Nrf2 protein was induced 2 h after LPS treatment (Figure 2B), it was completely inhibited by the treatment of CHX. Together, these results demonstrate that in response to LPS treatment, Nrf2 protein is synthesized de novo, rather than existing protein being liberated from Keap1. To determine if Nrf2 was regulated at the transcriptional level, we used the RNA synthesis inhibitor actinomycin D (ActD). Figure 2C shows that actinomycin D completely inhibited LPS-induced Nrf2 protein expression in THP-1 cells. Keap1 expression was unchanged in response to LPS treatment in THP-1 cells (Figure 2D), suggesting a minimal role for Keap1 in regulating LPS-induced Nrf2 activation. To confirm these results we also analyzed mRNA expression which unexpectedly showed that LPS induced the rapid expression of Nrf2 mRNA expression in THP-1 cells (Figure 2E). These results confirm that a third model of Nrf2 activation exists where Nrf2 transcription is increased, in this case by LPS treatment, which gives rise to transcribed Nrf2 mRNA and subsequent Nrf2 protein accumulation.
Nrf2 is a key transcription factor in the regulation of cytoprotective genes including NQO1 and HO-1. Nuclear accumulation is an important mechanism for the activation of Nrf2. We have recently shown that Nrf2 accumulates in the nucleus in response to LPS up to 4 h (16). After showing that Nrf2 expression is induced by LPS we next examined whether treatment with LPS induced Nrf2 nuclear accumulation beyond 4 h. THP-1 cells were treated with LPS for up to 12 h. Nuclear extracts were prepared and analyzed for Nrf2 expression by Western blot analysis. Figure 3A demonstrates that Nrf2 appeared in the nucleus in response to LPS with significant accumulation by 2 h and plateauing at around 8 h treatment. To verify these results in primary monocytes, we examined the subcellular localization of Nrf2 in response to LPS. Figure 3B shows that Nrf2 accumulates in the nucleus and cytosol by 2 h following LPS treatment in primary monocytes. The kinetics of Nrf2 protein appearance in the nucleus coincided with binding of a complex to the NQO1 ARE (Figure 3C). The binding of this complex was inhibited by the addition of unlabeled oligonucleotide, demonstrating specificity of the sequence (Figure 3D). The presence of Nrf2 in the complex was confirmed by supershift analysis. Anti-p65 (Rel A NF-κB) supershift antibodies were used as a negative control. The complex bound to the NQO1 ARE was abolished with anti-Nrf2 antibodies but not with anti-p65 antibodies (Figure 3D).
Since it has been shown that Keap1 regulates the redox sensitive activation of Nrf2 (26), and that ROS is known to be produced in response to LPS (32), the role of ROS was examined in this system. We analyzed the production of ROS in THP-1 cells using H2-DCFDA in response to LPS. In response to LPS, H2-DCFDA oxidation occurred in a time dependent manner (Figure 4A), suggesting that LPS induces ROS in THP-1 cells. The kinetics of ROS activation correlated with the nuclear accumulation of Nrf2 following LPS. However, the anti-oxidant N-acetyl cysteine (NAC) had no effect on LPS-induced Nrf2 accumulation in the cytosol and only minimally inhibited nuclear accumulation (Figure 4B) suggesting that the production of ROS by LPS is not required for Nrf2 activation.
The inflammatory responses induced by LPS are predominantly controlled through monocytes which produce the prototypic inflammatory cytokines TNF and IL-1β, and these in turn mediate many of the symptoms observed during sepsis (6,15). To understand the role of Nrf2 in modulating LPS-induced inflammation in human monocytes, Nrf2 siRNA was used to silence Nrf2 protein expression (Figure 5A). Primary human monocytes and THP-1 cells were either untransfected or transfected with control siRNA or with two independent Nrf2 targeted siRNA for 24 h. Western blot analysis of treated samples shows that both Nrf2 siRNA induced knockdown of the Nrf2 protein within 24 h. However, from this experiment Nrf2 siRNA 1 was deemed more effective than Nrf2 siRNA 2, and therefore this was used in subsequent experiments. To confirm that Nrf2 is responsible for the induced expression of NQO1 and HO-1 after treatment with LPS, cells were transfected with Nrf2 siRNA. Compared to control siRNA, Nrf2 siRNA 1 inhibited LPS-induced NQO1 and HO-1 mRNA expression (Figure 5B), verifying that Nrf2 regulates LPS-induced HO-1 and NQO1 expression in human monocytes. We then examined the role of Nrf2 in regulating LPS-induced TNF and IL-1β expression over a 48 h time period. Figure 5C shows that in response to LPS, there was a significant difference in expression levels of both TNF and IL-1β mRNA levels between control siRNA and Nrf2 siRNA samples from 16 h up to and including the 32 h time point. These results suggest that Nrf2 inhibits prolonged LPS-induced pro-inflammatory responses and not the initial transient LPS-induced responses.
Both HO-1 and NQO1 have been shown to exert anti-inflammatory properties (11,34). To determine if the effects seen with Nrf2 siRNA on LPS-induced TNF and IL-1β, in Figure 5, are due to the induced expression of HO-1 and NQO1, we used siRNA to silence HO-1 and NQO1 in isolation and also in combination. Transfection of THP-1 cells with HO-1 siRNA and NQO1 siRNA alone or in combination resulted in almost complete knockdown of their respective protein expression (Figure 6A). Furthermore, Figure 6B shows that LPS-induced HO-1 and NQO1 mRNA expression is significantly and specifically reduced in cells transfected with either HO-1 siRNA, NQO1 siRNA or both. Next we examined the effect of HO-1 and NQO1 siRNA on LPS-induced TNF and IL-1β mRNA expression. Figure 6C shows that HO-1 and NQO1 siRNA alone significantly prolonged the LPS-induced TNF and IL1β expression. In addition, when we used HO-1 and NQO1 siRNA in combination a more significant increase in TNF and IL-1β expression resulted from the LPS treatment at any time point, when compared to control siRNA alone. Similarly, LPS-stimulated THP-1 cells transfected with siRNA targeted to Nrf2, HO-1 and NQO1 showed greater TNF protein expression when compared to control siRNA transfected cells (Figure 6D). In contrast, silencing HO-1, NQO1 or Nrf2 had no effect on the activation of the anti-inflammatory cytokine interleukin 10 in THP-1 cells (data not shown). Taken together, these results demonstrate that both HO-1 and NQO1 play an important role in regulating the magnitude of LPS-induced pro-inflammatory responses in human monocytes.
NF-κB is known to regulate the expression of several genes that are essential for initiating and promoting inflammation, including TNF and IL-1β (15). Since silencing Nrf2, HO-1 and NQO1 in monocytes enhanced LPS-induced pro-inflammatory gene expression, their effects on NF-κB activation were then studied. Following LPS stimulation, NF-κB activation was examined 30 min after LPS stimulation in THP-1 cells using a p65 DNA-binding assay. Silencing HO-1 or Nrf2 resulted in significantly enhanced LPS-induced p65 DNA-binding, suggesting an inhibitory role of Nrf2 and HO-1 on NF-κB activation (Figure 6E). Interestingly, silencing NQO1 did not affect LPS-induced p65 DNA-binding, suggesting that the anti-inflammatory effects of NQO1 are NF-κB–independent.
To confirm that HO-1 and NQO1 inhibit LPS-induced TNF and IL-1β expression, we overexpressed HO-1 and NQO1 in THP-1 cells and then examined these cells for IL-1β and TNF expression in response to LPS. Figure 7A demonstrates that overexpression of pHO-1 or pNQO1 in THP-1 cells resulted in their respective protein expression. Figure 7B shows the expression of TNF and IL-1β in response to LPS in cells overexpressing either HO-1 alone, NQO1 alone, or HO-1 and NQO1 in combination. Transfection with pCDNA3.1 vector alone was used as a control. Results demonstrated that LPS-induced TNF and IL-1β were significantly inhibited by either HO-1 alone or NQO1 alone. Moreover, when used in combination, HO-1 and NQO1 overexpression had an additive effect on the inhibition of LPS-induced TNF and IL-1β expression. Taken together, these results demonstrate that HO-1 and NQO1 alone and in combination modulate LPS-induced pro-inflammatory responses.
The dietary anti-oxidant sulforaphane has previously been shown to activate the Nrf2 pathway (10). To determine if sulforaphane can induce HO-1 and NQO1 expression in human monocytes, THP-1 cells were treated with sulforaphane for up to 8 h. Figure 8A and 8B demonstrate that sulforaphane induced NQO1 and HO-1 mRNA and protein expression in a time-dependent manner. To establish if sulforaphane protects against LPS-induced inflammatory responses, THP-1 cells were pre-treated with sulforaphane for 4 h to induce HO-1 and NQO1 expression prior to activation with LPS for 4 h. Figure 8C demonstrates that sulforaphane inhibited LPS-induced TNF and IL-1β mRNA expression. To determine if NF-κB was involved in this pathway, the effect of sulforaphane on LPS induced NF-κB DNA-binding was examined. Sulforaphane suppressed LPS-induced p65 DNA-binding (Figure 8D). Taken together, these results suggest that pre-activating the Nrf2 pathway with sulforaphane inhibits LPS-induced pro-inflammatory responses in monocytic cells.
Understanding host responses that modulate inflammation during sepsis is key to developing new therapeutic strategies to reduce the increasing incidence of this complex disease in developed countries. The present study demonstrates that Nrf2 induces the phase II enzyme NQO1 which in combination with HO-1 modulates LPS induced inflammatory responses in human monocytes. Most research to date regarding the importance of Nrf2 in regulating inflammatory responses has focused on rodent models and the induction of the cytoprotective gene HO-1 (12,17,18). This is the first time inducible NQO1 expression has been shown to modulate IL-1β and TNF up-regulation.
The regulation of the phase II enzyme NQO1 in monocytes in response to LPS and other pro-inflammatory stimuli has not been studied. In this report gene expression profiling demonstrated that LPS unexpectedly induced the expression of NQO1 in primary monocytes and THP-1 cells. The expression of other Nrf2 regulated genes including GCLM GCLC, GSTA1, GSTA2, TrxR1 and GR were examined in response to LPS. No significant induction was observed in any of the genes associated with glutathione synthesis. This is an important finding since the glutathione system has been linked with regulating airway inflammation in mice (35).
Since NQO1 has been shown to be regulated by Nrf2, AP-1 and NF-κB (36-38), and because all of these transcription factors are known to be activated in response to LPS in human monocytes (16,39), we examined the mechanism by which LPS induced NQO1 expression. We found that LPS-induced NQO1 expression was regulated by Nrf2. Monocytes therefore induce the expression of both HO-1 and NQO1 in response to LPS through the activation of Nrf2. This suggests that Nrf2 mediates its anti-inflammatory effects by inducing the expression of NQO1, in addition to HO-1, whose role it is to protect monocytes against excessive activation.
NQO1 has been shown to have both anti-inflammatory and pro-inflammatory properties in different cell systems (11,40,41). Jaiswal and co-workers have recently documented that NQO1 plays an important role in TNF-induced NF-κB activation (40), in which NQO1 is thought to modulate NF-κB through its control of redox status. In contrast, other groups have shown that NQO1 has anti-inflammatory properties. Chen et al (2003) describes how NQO1 can inhibit TNF-induced VCAM-1 expression in human endothelial cells (11). Interestingly, it was reported that this regulation of TNF- induced VCAM-1 expression was not through NF-κB inhibition (11,41). Thimmulappa et al (2007) reported that CDDO-Im (a synthetic triterpenoid) induces NQO1 expression in neutrophils, which subsequently inhibited LPS-induced IL-6 and TNF induction (13). The results of the current study show that overexpression of NQO1 alone as well as in combination with HO-1 can inhibit LPS-induced TNF and IL-1β expression in human THP-1 cells and this inhibition is NF-κB-independent. Taken together, these studies suggest a multifunctional role for NQO1 in modulating inflammation.
We have previously shown that LPS induces HO-1 in human monocytes through the activation of Nrf2 (16). In this study we examined the function of this induction by targeting HO-1 for gene silencing as well as overexpression studies. The results demonstrated that by silencing HO-1 expression an excessive inflammatory response in human monocytes is induced in response to LPS. Moreover, when we overexpressed HO-1 we observed that the inflammatory response induced by LPS was inhibited. Furthermore, when combining HO-1 with NQO1 silencing (Figure 5), we observed an increase in LPS-induced TNF and IL-1β expression above silencing HO-1 or NQO1 alone. In addition, when we overexpressed a combination of both NQO1 and HO-1 we observed a significant decrease in LPS-induced TNF and IL-1β expression. When we silenced Nrf2 we detected a similar response to that seen with NQO1 and HO-1 silenced together. These results taken together suggest that Nrf2 mediates its anti-inflammatory effects by inducing the expression of HO-1 and NQO1, which in turn regulate the strength of the inflammatory response in human monocytes.
Studies have shown that HO-1 plays an important role in modulating inflammatory responses (42-44). Many anti-inflammatory mediators including IL-10, and 15-deoxy-prostaglandin J2 have been shown to up-regulate HO-1 which subsequently inhibits inflammation (44,45). The anti-inflammatory effects of HO-1 are thought to be mediated by its metabolites carbon monoxide and bilirubin, which have been reported to inhibit NF-κB activation in various cells and tissues (44,45). Here, we found that overexpression of HO-1 and NQO1 can inhibit LPS-induced TNF and IL-1β expression. However, HO-1 inhibits LPS-induced TNF and IL-1β expression through suppression of NF-κB, while NQO1 inhibits this response through an NF-κB-independent pathway. Further work is required to establish this important mechanistic difference as we may be able to use this diversity between HO-1 and NQO1 in inhibiting inflammatory responses to our advantage in a therapeutic setting.
The mechanism by which Nrf2 becomes activated in response to its many inducers is a matter of much discussion (22-27). This report adds a new insight in to that discussion since we have observed that upon activation with LPS, Nrf2 mRNA is synthesized de novo and is not dependent on production of ROS. Nrf2 protein is subsequently transcribed and functions to induce NQO1 and HO-1 expression. This suggests that Keap1 is either redundant in this system or it is there to stabilize the transcribed Nrf2 protein, before it moves into the nucleus. This raises a number of different questions, firstly, what transcription machinery controls Nrf2 mRNA induced expression? And secondly can we target this transcriptional machinery for therapeutic intervention. These are questions that need to be addressed as this information may help provide novel therapies for the treatment of sepsis as well as other diseases associated with Nrf2.
The results of the current study show that in human monocytes up-regulation of NQO1 and HO-1 by LPS is dependent on Nrf2. Furthermore, we report that LPS-induced Nrf2 activation protects monocytes from excessive inflammatory responses. Results were similar in primary monocytes and THP-1 cells, indicating that THP-1 cells are a valid model for studying Nrf2 activation as well as NQO1 and HO-1 gene expression in human monocytes. Nrf2 deficiency results in increased mortality from LPS in experimental models of sepsis. In addition, we have shown here that the Nrf2-regulated genes NQO1 and HO-1 protect LPS-activated monocytes from excessive inflammatory responses. Moreover, we also describe a novel mechanism for the activation of Nrf2 by LPS (Figure 9). Taken together, further understanding of the transcriptional machinery that regulates Nrf2 and its gene products may provide a novel therapeutic target for the treatment of sepsis.
The authors thank Dr D Sexton (University of East Anglia) for the TNFα ELISA.