TNF Mediates the Sustained Activation of Nrf2 in Human Monocytes

Monocytes are the main producers of TNF in innate immune responses and in chronic inflammatory diseases such as rheumatoid arthritis. Investigations of the actions of TNF have mostly focused on acute activation of other cell types such as endothelial cells (1). Less is known about the effects of TNF on monocytes themselves, and little is known about the regulation of cell responses to TNF beyond the initial acute and transient activation of signaling pathways and transcription factors such as NF-κB. Studies have emphasized the importance of mechanisms and autocrine loops that sustain and regulate the activity of signaling pathways and transcription factors after the initial and typically transient response to an extracellular ligand (2).

TNF is a pleiotropic cytokine that regulates a broad range of biological activities, including cell differentiation, proliferation, and death, as well as inflammation, innate and adaptive immune responses, and tissue development (3). TNF binds to two distinct receptors on the cell surface, TNFR1 and TNFR2 (4). TNFR1 is constitutively expressed by most cell types, and TNFR2 expression is mainly in immune cells. Most of the biological effects of TNF are attributed to TNFR1, but there is also evidence that both receptors transduce signals cooperatively (5, 6). Ligation of both receptors results in the activation of downstream kinase pathways, NF-κB and the AP-1 transcriptional activator (6). Cellular responses to TNF vary depending on the cell type and microenvironment and are controlled by the balance of activation of various signaling pathways (6, 7).

Recently, the transcription factor NF-E2–related factor 2 (Nrf2) has emerged as a key regulator of inflammation (8, 9). The main role of Nrf2 is to protect cells from oxidative damage by inducing the expression of various cytoprotective and detoxification genes (10). The genes regulated by Nrf2 include the cytoprotective enzyme heme oxygenase-1 (HO-1), as well as the phase II enzymes such as NAD(P)H:quinone oxidoreductase 1 (NQO1) and GSTα1 (GSTA1), which detoxify endogenous and exogenous chemicals through reduction and conjugation reactions (10). It also includes the regulation of glutamyl cysteine ligase modulatory (GCLM) and glutamyl cysteine ligase catalytic (GCLC) units, the two subunits of the rate-limiting enzyme in glutathione biosynthesis. Several of these Nrf2-regulated genes have been shown to have anti-inflammatory actions (8, 9, 11–15), which suggests that Nrf2 mediates its control on inflammation by inducing the expression of any number of ARE-dependent genes.

In this study, we report that TNF induces prolonged Nrf2 transcriptional activation in human monocytes. We demonstrate that TNF induced autocrine TNF upregulation, which was responsible for prolonged activation of Nrf2. Previously, we have shown that LPS can induce the expression of HO-1 and NQO1 in human monocytes by an Nrf2-dependent mechanism (8). In this study, we show that TNF can mediate the upregulation of NQO1, GCLM, and GSTA1, but not HO-1. We show that silencing TNF-induced Nrf2 responses leads to a greater induction of proinflammatory cytokines mediated by NF-κB. We also show that TNF induces autocrine TNF expression that further prolongs Nrf2 activation and cytoprotective gene induction. These data suggest that a functional autocrine loop is evident in human monocytes in response to TNF, thus providing new insight into the regulation of inflammatory processes regulated by human monocytes.

All Abs were obtained from Santa Cruz Biotechnology (Santa Cruz, CA). Control Nrf2, Bach1 p50, and p65 small interfering RNA (siRNA) were purchased from Applied Biosystems (Foster City, CA). All other reagents were obtained from Sigma-Aldrich (St. Louis, MO), unless indicated.

Primary human monocytes were cultured in RPMI 1640 medium supplemented with 10% FCS and 2 mM l-glutamine (Invitrogen). 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 Histopaque (Sigma-Aldrich) density gradient centrifugation. PBMCs (4 × 10⁶/ml) were incubated in complete medium for 2 h at 37°C to allow adherence of monocytes (16). Cell type was confirmed by microscopy and flow cytometry.

Total RNA was extracted from 5 × 10⁵ 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 were purchased from Invitrogen. Sequences of primers were as follows: GAPDH, forward 5′-ACCAGCCTCAAGATCATCAGC-3′ and reverse 5′-TGCTAAGCAGTTGGTGGTGC-3′; HO−1, forward 5′-ATGGCCTCCCTGTACCACATC-3′ and reverse 5′-TGTTGCGCTCAATCTCCTCCT-3′; Nrf2, forward 5′-AAACCAGTGGATCTGCCAAC-3′ and reverse 5′-AGCATCTGATTTGGGAATGTG-3′; GCLM, forward 5′-GCGAGGAGCTTCATGATTGT-3′ and reverse 5′-CTGGAAACTCCCTGACCAAA-3′; NQO1, forward 5′-GCCGCAGACCTTGTGATATT-3′ and reverse 5′-TTTCAGAATGGCAGGGACTC-3′; GCLC, forward 5′- GGCGATGAGGTGGAATACAT-3′ and reverse 5′-GTCCTTTCCCCCTTCTCTTG-3′; ferritin, forward 5′-CTGGAGCTCTACGCCTCCTA-3′ and reverse 5′- TGGTTCTGCAGCTTCATCAG-3′; IL-1β, forward 5′-AGCTGATGGCCCTAAACAGA-3′ and reverse 5′-GGAGATTCGTAGCTGGATGC-3′; and TNF, forward 5′-GCCCAGGCAGTCAGATCATC-3′ and reverse 5′-CGGTTCAGCCACTGGAGCT-3′.

Relative quantitative real-time PCR used SYBR Green technology (Roche) on cDNA generated from the reverse transcription of purified RNA. After preamplification (95°C for 2 min), the PCRs were amplified for 45 cycles (95°C for 15 s, 60°C for 10 s, and 72°C for 10 s) on a LightCycler 480 (Roche). Each mRNA expression was normalized against GAPDH mRNA expression using the standard curve method.

NaDodSO₄-PAGE and Western analyses were performed, as described previously (7). Briefly, whole-cell lysates were extracted using radioimmunoprecipitation assay buffer method, and NaDodSO₄–PAGE separation was performed. Nuclear extracts were prepared, as previously described (17). Protein was transferred to nitrocellulose, and Western blot analysis was performed with the indicated antisera, according to their manufacturer’s guidelines. Flow cytometry for measuring apoptosis was performed on an Accuri-C6 flow cytometer. A dichlorofluorescein assay was used to determine cellular reactive oxygen species (ROS) generation in cells (18).

Primary monocytes (2 × 10⁶/well) were transfected using Amaxa Nucleofector Technology, using equivalent molar concentrations of the siRNA (to yield a final concentration of 30 nM). Transfected cells were incubated for either 24 or 48 h before the indicated treatments.

NF-κB DNA binding was measured using the NF-κB p65 and P50 transcription factor ELISA kit (Panomics).

Student t test was performed to assess statistical significance from controls. Results with p values <0.05 were considered statistically significant (*). Results represent the mean ± SEM of three independent experiments. For Western blotting experiments, data are representative of three independent experiments.

Nrf2 is a key transcription factor in the regulation of antioxidant and cytoprotective genes, and through these genes we have previously shown that Nrf2 can regulate LPS-induced inflammatory processes in human monocytes (8). Our group’s focus is examining TNF-regulated signaling responses in human inflammatory cells with a view to understanding the basic mechanisms that control inflammatory responses in normal and disease states. To address this focus, we first examined the expression of Nrf2 in response to TNF in human monocytes. Fig. 1A shows that TNF can induce Nrf2 protein expression in human monocytes, and that this expression is stable up to and including 72 h. This Western blot for Nrf2 expression shows two bands at ∼65 kDa and 110 kDa. These two bands equate to Nrf2 protein (∼65 kDa) and ubiquitinated Nrf2 (∼110 kDa). Both bands show an increase in response to TNF in human monocytes. We also examined monocyte viability by measuring apoptosis in response to TNF for 72 h. Supplemental Fig. 1A shows that no difference was apparent between the different treatment groups. Cytosolic and nuclear expression is an important marker for the activation of Nrf2. To determine whether TNF can induce Nrf2 nuclear expression, we examined the nuclear and cytosolic expression of Nrf2 in response to TNF. Fig. 1B shows that TNF induces nuclear accumulation of Nrf2 in human monocytes. Next, we examined Nrf2-regulated genes in response to TNF in human monocytes. We examined the mRNA expression of ferritin, GCLM, GCLC, HO-1, NQO1, and GSTA1 in response to TNF stimulation at 2, 24, and 72 h. TNF significantly increased NQO1, GSTA1, and GCLM mRNA expression in primary human monocytes (Fig. 1C). Unexpectedly, TNF stimulation did not induce HO-1 mRNA expression, but slightly reduced its expression. To confirm that Nrf2 was responsible for the induction of TNF-induced NQO1, GSTA1, and GCLM, we used Nrf2 siRNA to inhibit its function. Fig. 2A shows that we can effectively knock down Nrf2 in human monocytes. Fig. 2B shows that Nrf2 siRNA blocked TNF-induced NQO1, GCLM, and GSTA1 expression in human monocytes. We next examined monocyte viability in Nrf2-silenced monocytes compared with control cells in response to TNF for 24 h. Supplemental Fig. 1B shows a slight decrease in monocyte viability in those cells treated with Nrf2 siRNA in combination with TNF when compared with control cells.

Because it has been shown that Keap1 regulates the redox-sensitive activation of Nrf2 (19), and that ROS is known to be produced in response to TNF (20), the role of ROS was examined in regulating TNF-induced Nrf2 activation. We analyzed the production of ROS in monocytes with and without Nrf2 siRNA using dichlorodihydrofluorescein diacetate (H₂DCFDA) in response to TNF. In response to TNF, H₂DCFDA oxidation occurred in a time-dependent manner (Fig. 2C), suggesting that TNF induces ROS in monocytes. Furthermore, when we silence Nrf2, we get an increase in ROS production compared with control siRNA, when treated with TNF. The kinetics of ROS activation correlated with the nuclear accumulation of Nrf2 following TNF. The antioxidant N-acetyl cysteine (NAC) inhibited TNF-induced Nrf2 induction (Fig. 2D) at 4 and 24 h, suggesting that the production of ROS and the activation of Keap1 are required for the sustained TNF-induced Nrf2 activation in monocytes.

Next, we wanted to address the functional importance of TNF-induced Nrf2-mediated gene expression, as well as examining the different kinetics of Nrf2-mediated gene expression to TNF treatment. First, we wanted to determine whether the pattern of TNF-induced Nrf2-mediated gene expression was induced by TNF and not by contaminating microbial products. We prevented binding of TNF to its receptor using soluble TNF receptor (Humira). Fig. 3A and 3B shows that Nrf2 nuclear expression and Nrf2-mediated gene expression are attenuated with the cotreatment of TNF and anti-TNF (Humira).

It is important to establish the functional relevance of TNFR1 and TNFR2 in regulating TNF-mediated cytoprotective gene expression in human monocytes. Next, we used siRNA to knock down TNFR1 and TNFR2 (Fig. 3C). Targeting mRNA knockdown for TNFR1 and TNFR2 shows that inhibiting the expression of TNFR1 alone or in combination with TNFR2 blocks the TNF-induced Nrf2-mediated activation (Fig. 3D). Fig. 3E confirms that TNFR1 alone or in combination with TNFR2 blocks the TNF-induced Nrf2-mediated gene upregulation.

To understand the kinetics of prolonged autocrine TNF responses, we first examined the mRNA expression of TNF to TNF treatment in human monocytes over a 48-h time period with a 1-h pretreatment or 2-h posttreatment with anti-TNF. Fig. 4A shows that TNF mRNA doesn’t return to basal levels even after 48 h. When we preincubated with anti-TNF for 1 h, prior to TNF treatment, unsurprisingly no TNF mRNA was induced. When we added anti-TNF after 2 h of TNF treatment, this caused TNF mRNA levels to return to constitutive levels within 12 h. Next, we examined Nrf2 nuclear expression in response to TNF with a 1-h pretreatment or 2-h posttreatment with anti-TNF. Fig. 4B showed that preincubation with anti-TNF, followed by TNF treatment, prevented Nrf2 nuclear localization. When we added anti-TNF after 2 h of TNF treatment, this caused Nrf2 nuclear levels to be elevated by 4 h, but return to constitutive levels by 24 h. Next, we examined the mRNA expression of Nrf2-regulated genes in response to TNF in combination with anti-TNF treatment. Fig. 4C shows that when we preincubated with anti-TNF for 1 h, prior to TNF treatment for 24 h, this blocked Nrf2-induced gene expression. However, when we added anti-TNF after 2 h of TNF treatment, this caused no significant reduction in the mRNA expression of GCLM or GSTA1, but NQO1 was significantly reduced by this treatment. Finally, we wanted to determine whether NF-κB was needed for the regulation of sustained TNF-induced Nrf2 nuclear expression. Fig. 4D shows that when we add the inhibitor of NF-κB, BAY 11-7082, in combination with TNF, we observe an inhibitory effect after 24 h of TNF treatment, but not at 4 h. Fig. 4D also shows a similar response to the mRNA of the Nrf2-regulated gene NQO1. We next examined monocyte viability in BAY 11-7082–treated monocytes compared with control cells in response to 24-h TNF treatment. Supplemental Fig. 1B shows that a significant decrease in monocyte viability exists in cells treated with BAY 11-7082 plus TNF when compared with control cells. This result is in line with previously published data (21).

To understand the significance of TNF-induced Nrf2 activation in human monocytes, we used siRNA to target knockdown of Nrf2. Fig. 2A shows that we can effectively and specifically knock down mRNA levels of Nrf2 in human monocytes. Next, we examined the effect of Nrf2 siRNA on TNF-induced TNF and IL-1β mRNA expression. Fig. 5A shows that Nrf2 siRNA significantly prolonged the TNF-induced TNF and IL-1β expression 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. Because silencing Nrf2 in monocytes enhanced TNF-induced proinflammatory gene expression, their effects on NF-κB activation were then studied. Following TNF stimulation, NF-κB activation was examined after TNF stimulation in human monocyte cells using a p50 and p65 DNA-binding assay. Silencing Nrf2 resulted in significantly enhanced TNF-induced p50 and p65 DNA binding, suggesting an inhibitory role of Nrf2 on NF-κB activation (Fig. 5B).

Studies have emphasized the importance of mechanisms and autocrine loops that sustain and regulate the activity of signaling pathways and transcription factors after the initial and typically transient response to an extracellular ligand (2, 22). Research is limited in the study of TNF on monocytes, that is, not much is known about the regulation of monocyte responses to TNF beyond the initial acute and transient activation of signaling pathways (23, 24). The present study aimed to address the long-term exposure of monocytes to TNF. We showed that TNF induces the expression and activation of the transcription factor Nrf2, which leads to the upregulation of cytoprotective and detoxification genes, including NQO1, GCLM, and GSTA1. The regulation of such genes by Nrf2 has been shown to regulate innate immune responses in various experimental disease models (25, 26). This is the first time, to our knowledge, that TNF alone has been shown to regulate the expression and activation of Nrf2.

The control of Nrf2 transcription and its subsequent transcriptional responsibilities in controlling NQO1, GCLM, and GSTA1 is required for the dampening of TNF-induced inflammatory responses, without which inflammation could and probably would run unchecked. For example, NQO1 has been shown to be anti-inflammatory by modulating NF-κB (27). Moreover, other groups have shown that NQO1 is anti-inflammatory and can inhibit TNF-induced VCAM-1 expression in human endothelial cells (28). Interestingly, it was reported that this regulation of TNF-induced VCAM-1 expression was not through NF-κB inhibition. Thimmulappa et al. (9) reported that CDDO-Im (a synthetic triterpenoid) induces a number genes, including NQO1 in neutrophils, and was associated (coincided) with the inhibition of LPS-induced IL-6 and TNF induction. The results of the current study show that TNF-induced expression of NQO1 in combination with other Nrf2-regulated genes can inhibit TNF-induced TNF and IL-1β expression in human monocytes, and that this inhibition seems to be sustained and NF-κB dependent. Our results also suggest that Nrf2-regulated glutathione-induced signaling may also play an essential role in regulating immune response in human monocytes.

The present study demonstrates that ROS regulates the activation of Nrf2 in response to treatment with TNF; however, what is the significance of increased Nrf2-targeted antioxidant and detoxification genes in this system? One could argue that increasing antioxidant genes is to counteract increased ROS caused by TNF treatment, and thus protect monocytes from undergoing ROS-induced apoptosis. In this study, we showed a decrease in monocytes’ viability in response to silencing of Nrf2 in combination with TNF treatment. This is an important physiological finding and would certainly be beneficial to an inflammatory process in which monocytes are important mediators of such responses. However, if Nrf2 signaling is interrupted, then inflammation may well run unchecked and result in the progression to inflammation-associated diseases.

In this study, we also showed that in human monocytes, HO-1 mRNA expression is reduced in response to TNF. These results corroborate work by Kirino et al. (29), who showed that TNF induces HO-1 downregulation in human monocytes by promoting the degradation of HO-1 mRNA. In our laboratory, we have shown that under normal conditions, human monocytes express significantly more HO-1 than other inflammatory cells (S.A. Rushworth and D.J. MacEwan, unpublished observations). This begs the question as to why these cells regulate this gene in such an aggressive way. We can certainly speculate that to get a significant inflammatory response to TNF, monocytes have to first control the expression of anti-inflammatory genes. However, if this is the case, then why does TNF not promote degradation of other protective genes? This concept that TNF can promote HO-1 mRNA degradation is very interesting and needs further investigation.

Another interesting finding of this study is that when we block TNFR signaling or oxidant activity using NAC, this leads to inhibition of Nrf2 nuclear accumulation in monocytes. However, when we inhibit NF-κB using BAY 11-7082, this does not lead to inhibition of Nrf2 nuclear accumulation. This suggests that induction of ROS and subsequently Nrf2 nuclear accumulation in monocytes are independent of NF-κB activation. Moreover, blocking NF-κB activity alone or in combination with TNF has been shown to increase the levels of ROS, which can induce apoptosis (21, 30, 31). In this study, no apoptosis was observed in human monocytes by the treatment of NF-κB inhibition. Taken together, these results indicate that NF-κB does not play a role in regulating early Nrf2 nuclear accumulation in human monocytes.

We have recently shown that LPS can induce Nrf2 expression and activation in human monocytes; moreover, we also show that HO-1 is induced in this system (8). This raises an important point for discussion, which is why does TNF have little effect on HO-1 expression, whereas LPS induces its induction within the same cells? The answer to that question may lie in the mechanism of HO-1 regulation. This is because the control of HO-1 gene is under the control of not just Nrf2, but many other transcription factors (30, 32–35). Among the transcription factors NF-κB, Nrf2, and AP-1, families are arguably the most important, and among the best studied; however, others, including heat shock factor, upstream stimulatory factors, Ets-2, hepatocyte NF-4 with Sp1 or Sp3, and STAT proteins, have all been implicated in regulating HO-1 transcription (34).

In conclusion, the results of the current study show human monocytes depleted of Nrf2 have elevated levels of TNF-induced inflammatory cytokines, which may promote their recruitment of other leukocytes, thereby perpetuating inflammation and injury in response to infection. Furthermore, we show that TNF regulates the activation of the transcription factor Nrf2 and its associated gene targets. This occurs through a TNFR1-dependent mechanism with autocrine TNF production sustaining the Nrf2 activation up to 72 h posttreatment. Taken together, understanding of these mechanisms that protect against excessive inflammatory responses may pave the way to provide novel therapeutic strategies for the treatment of inflammation-associated disease processes.

The authors have no financial conflicts of interest.