Increasing evidence demonstrates that melatonin regulates inflammatory and immune processes acting as both an activator and inhibitor of these responses. Nevertheless, the molecular mechanisms of its anti-inflammatory action remain unclear. Here we have characterized the cellular mechanisms underlying the redox modulation of LPS-stimulated inflammatory responses in murine peritoneal macrophages by melatonin to provide insight into its anti-inflammatory effects.
Murine peritoneal macrophages were isolated and treated with melatonin in the presence or absence of LPS (5μg·mL−1) for 18 h. Cell viability was determined using sulforhodamine B assay and NO production was measured using the Griess reaction. Pro-inflammatory enzymes and transcription factors were detected by Western blotting.
Without affecting cell viability, melatonin (12.5, 25, 50 and 100 μM) reduced the level of nitrites, inducible NOS (iNOS), COX-2 and microsomal PGE synthase-1 (mPGES1) protein, and p38 MAPK phosphorylation, and prevented NF-κB translocation. Furthermore, melatonin treatment significantly increased NF-E2-related factor 2 (Nrf2) and haem oxygenase 1 (HO1) protein levels in murine macrophages exposed to LPS.
Conclusions and Implications
Melatonin reduced pro-inflammatory mediators and enhanced the expression of HO1 via NF-κB, p38 MAPK and Nrf2 cascade signalling pathways in murine macrophages. Thus, melatonin might be a promising target for diseases associated with overactivation of macrophages.
Melatonin (N-acetyl-5-methoxytryptamine), is a neurohormone synthesized from the aromatic amino acid tryptophan mainly by the pineal gland of mammals (Reiter, 1991) and other extrapineal organs and tissues including skin (Slominski et al., 2002), retina (Faillace et al., 1995), harderian gland, gastrointestinal tract (Huether et al., 1992; Bubenik, 2002), ovary, testes, bone marrow (Tan et al., 1999), thymus, spleen (Sánchez-Hidalgo et al., 2009) and in leukocytes (Carrillo-Vico et al., 2004). The biological functions of melatonin have been investigated extensively. For instance, melatonin regulates seasonal reproduction and circadian rhythm (Reiter et al., 2011). This indolamine also acts as a powerful and widely effective antioxidant, as it has been shown to scavenge different types of free radicals in vitro and in vivo (Allegra et al., 2003; Reiter et al., 2009; du Plessis et al., 2010; Tamura et al., 2013) and to activate antioxidant defences such as superoxide dismutase (SOD), catalase, GSH peroxidase, GSH reductase and glucose-6-phosphate dehydrogenase (De La Lastra et al., 1997; Alarcón de la Lastra et al., 1999; Hardeland, 2009), consequently reducing oxidative stress. Likewise, a large number of reports describe melatonin as an immunomodulatory compound acting on specific receptors in immunocompetent cells (Guerrero and Reiter, 2002). Nevertheless, it still remains unclear how melatonin regulates immunity. In this context, while some authors argue that melatonin is an immunostimulant, many other studies have described its anti-inflammatory properties (Carrillo-Vico et al.,2013).
In experimental in vivo and in vitro inflammation, melatonin modulated arachidonic acid metabolism, preventing or reducing the inflammatory activation of PLA2, lipoxygenase and COX-2 (Radogna et al., 2010). According to recent studies, melatonin suppressed the production of NO and IL-6 at both gene transcription and translation levels in LPS-activated macrophages (Choi et al., 2011). Moreover, melatonin might modulate Toll-like receptor 4-mediated inflammatory genes through MyD88- and TRIF-dependent signalling pathways in LPS-stimulated RAW264.7 macrophages (Xia et al., 2012). Nevertheless, the intracellular molecular mechanisms involved in melatonin effects in inflammation remain, at least in part, unclear and need to be explored in depth.
Immunocompetent cells that have melatonin receptors are target cells for its immunomodulatory function (Carrillo-Vico et al., 2003) cooperating during the onset, progression and resolution of inflammation (Soehnlein and Lindbom, 2010). In addition, melatonin exerts an important role in managing inflammatory responses, modulating the ability of endothelial cells to control the rolling, adhesion and transmigration of leukocytes through blockade of NF–κB-dependent mechanisms (Marçola et al., 2013). In fact, the circadian rhythm of melatonin primes the ability of endothelial cells to adhere to neutrophils in the day whereas, at night, melatonin in the blood maintains endothelial cells in a low reactive state.
Macrophages play a critical role in inflammation. Resident macrophages produce cytokines and chemokines that attract other cells, including neutrophils and additional macrophages. All of these responses can be used as readouts and are useful in assessing the role of pathogenic genes or proteins (Schneider, 2013). Murine and human macrophages exhibit a particularly vigorous response to LPS, which induces a variety of inflammatory modulators (Adams and Hamilton, 1984). LPS stimulation of macrophages disrupts the balance of the intracellular redox state, which leads to oxidative stress characterized by a major shift in the cellular redox balance and is usually accompanied by damage mediated by reactive oxygen species (ROS) (Brüne et al., 2013). Macrophages express enzymes such as inducible NOS (iNOS) and COX-2 that regulate inflammatory processes (Chang et al., 2012) and these proteins are responsible for the overproduction of NO and PGE2, respectively, during inflammation. Apart from these enzymes, production of another inflammatory mediator PGE2 is triggered by activation of microsomal PGE synthase-1 (mPGES1), an efficient downstream enzyme co-localized and functionally coupled with COX-2 in macrophages activated by LPS (Lazarus et al., 2002). The process of gene expression of these pro-inflammatory mediators involves several signal transduction pathways such as the MAPK and NF-κB pathways (Barton and Medzhitov, 2003; Qi and Shelhamer, 2005). Importantly, a key transcription factor, NF-E2-related factor-2 (Nrf2), is an orchestrator of the induction of several antioxidant enzymes, such as haem oxygenase 1 (HO1; nomenclature follows Alexander et al., 2013) and thus regulates the cellular antioxidant response against ROS in murine macrophages, modulating acute inflammatory responses (Jung et al., 2010a; Kang and Kim, 2013). These pro-inflammatory mediators and pathways are regarded as essential anti-inflammatory targets (Lawrence et al.,2002). For this reason, the stimulation of macrophages with LPS constitutes an excellent model for the screening and subsequent evaluation of the effects of candidate drugs on the inflammatory pathway (Sánchez-Miranda et al., 2013).
Taking this background into account, the aim of the present study was to address the intracellular mechanisms underlying the effects of melatonin on the inflammatory responses induced by LPS, in murine macrophages. In this model, redox changes, protein expression of pro-inflammatory (iNOS, mPGES1, COX-2) and anti-inflammatory (HO1) enzymes, along with the roles of MAPK, NF-κB and Nrf2 signalling pathways involved in melatonin effects after the induction of inflammation were also determined.
All animal care and experimental procedures complied with the Guidelines of the European Union regarding animal experimentation (Directive of the European Counsel 86/609/EC) and followed a protocol approved by the Animal Ethics Committee of the University of Seville. All studies involving animals are reported in accordance with the ARRIVE guidelines for reporting experiments involving animals (Kilkenny et al., 2010; McGrath et al., 2010). Thirty 8–10-week-old male Swiss mice provided by Harlan Interfauna Ibérica® (Barcelona, Spain) weighing 20–30 g, were randomly placed in cages (five mice per cage) and maintained under constant conditions of temperature (20–25°C) and humidity (40–60%) with a 12 h light/dark cycle and fed standard rodent chow (Panlab A04, Seville, Spain) and water ad libitum throughout the experiment in our Animal Laboratory Center (Faculty of Pharmacy, University of Seville, Spain). After 1 week for acclimatisation, peritoneal macrophages were elicited by i. p. injection of 1 mL sterile thioglycollate medium (10% w/v; Scharlau®, Barcelona, Spain), from five mice. Following the thioglycollate injection, mouse behaviour, water and food consumption, loss of body weight and survival were monitored daily until killing, 3 days later (see below).
Isolation and culture of murine peritoneal macrophages
Mice were injected intraperitoneally with 1 mL of sterile thioglycollate medium (10% w/v). After 3 days, mice were killed by CO2 inhalation, and peritoneal exudate cells were harvested by washing the peritoneal cavity with sterile ice-cold PBS (Alleva et al., 2002). After centrifugation, cells were resuspended in RPMI 1640 medium (PAA®, Pasching, Austria) supplemented with 10% heat-inactivated fetal calf serum (FCS; PAA), l-glutamine (2 mM), glucose (4.5 g·L−1) and HEPES buffer (10 mM), in the presence of 100 mg·mL−1 streptomycin and 100 U·mL−1 penicillin (PAA) and then seeded in culture plates (1 × 106 cells·mL−1) for 2 h at 37°C in a 5% CO2-humidified atmosphere. After 2 h, non-adherent cells were removed by washing with PBS and fresh RPMI 1640 medium supplemented with 5% FCS containing the treatment was added. After 30 min, murine peritoneal macrophages (1 × 106 cells·mL−1) were treated with 5 μg·mL−1 of LPS from Escherichia coli (Sigma-Aldrich®, St Louis, MO, US) in absence or presence of melatonin (12.5, 25, 50 or 100 μM) for 18 h. In each experiment, viability was always ≥95%.
Cells seeded in 96-well plates (1 × 105 cells per well) were incubated in presence or absence of melatonin for 18 h. At the end of the exposure time, the effect of melatonin on cell growth/viability was analysed by sulforhodamine B (SRB) assay (Sigma-Aldrich; Skehan et al., 1990). After incubation time, adherent cell cultures were fixed in situ by adding 50 μL of 50% (w/v) cold of trichloroacetic acid (Sigma-Aldrich) and incubated for 60 min at 4°C. The supernatant was discarded and plates were washed five times with deionized water and dried. Fifty microlitres of SRB solution (0.4% w/v) in 1% acetic acid (Panreac) was added to each well and incubated for 30 min at room temperature. Plates containing SRB solution were washed five times with 1% acetic acid. Then, plates were air dried and 100 μL per well of 10 mmol·L−1 Tris base pH 10.5 (Sigma-Aldrich) were added and the absorbance of each well was read on an elisa reader at 510 nm (BioTek®, Bad Friedrichshall, Germany). Finally, cell survival was measured as the percentage of absorbance compared with that obtained in control cells (non-treated cells).
Measurement of nitrite production
Cells in 24-well plates were untreated or treated with different concentrations of melatonin (12.5, 25, 50 or 100 μM), and 30 min later stimulated with LPS for 18 h. The culture supernatants (100 μL) were transferred to a 96-well assay plate mixed with Griess reagent (Sigma) and incubated for 15 min at room temperature. The amount of nitrite, as an index of NO generation, was determined by a spectrophotometric method using the Griess reaction (Moorcroft et al., 2001) and obtained by extrapolation from a standard curve with sodium nitrite. The absorbance at 540 nm was measured by an elisa reader. Results were expressed as nitrite production, relative to LPS control cells (stimulated but untreated cells = 100%). Dexamethasone (1μM; Sigma) was used as positive control (data not shown).
Isolation of cytoplasmic and nuclear proteins and immunoblotting detection
Cells (1 × 106 cells·mL−1) were untreated or treated with melatonin and stimulated with LPS for 18 h. After incubation, cells were rinsed, scraped off and collected in ice-cold PBS containing a cocktail of protease and phosphatase inhibitors and processed as described by Sánchez-Hidalgo et al., (2005) in order to isolate cytoplasmatic or nuclear proteins. Protein concentration was measured for each sample using a protein assay reagent (Bio-Rad®, München, Germany) according to the Bradford's method and using ɣ-globulin as a standard (Bradford, 1976). Aliquots of supernatant containing equal amounts of protein (20 μg) were separated by SDS-PAGE on 10% acrylamide gel. In the next step, the proteins were electrophoretically transferred into a nitrocellulose membrane and incubated with specific primary antibodies: rabbit anti-COX-2 and rabbit anti-iNOS (Cayman®, Ann Arbor, MI, USA; 1:2500 and 1:1000, respectively), rabbit anti-mPGES1 (Cayman; 1:1000), rabbit anti-IκBα, (Cell Signalling®, Danvers, MA, USA; 1:1000), rabbit anti-p65, mouse anti-pJNK, rabbit anti-JNK, mouse anti-pp38, rabbit anti-p38 (Santa Cruz Biotechnology®, Santa Cruz, CA, USA; 1:1000), rabbit anti-HO1 and rabbit anti-Nrf2 (Santa Cruz Biotechnology; 1:500), overnight at 4°C. After rinsing, the membranes were incubated with a HRP-labelled secondary antibody anti-rabbit (Cayman Chemical; 1:50 000) or anti-mouse (Dako®, Atlanta, GA, USA; 1:2000) containing blocking solution for 1–2 h at room temperature. To prove equal loading, the blots were analysed for β-actin expression using an anti-β-actin antibody (Sigma Aldrich). Immunodetection was performed using enhanced chemiluminescence light-detecting kit (Pierce®, Rockford, IL, USA). The immunosignals were captured using LAS-3000 Imaging System from Fujifilm Image Reader (Stamford, USA) and densitometric data were studied following normalization to the house-keeping loading control. The signals were analysed and quantified by an image processing and analysis in Java (Image J, Softonic® Barcelona, Spain) and expressed in relation to the DMSO–LPS-treated cells.
All values in the Figures and text are expressed as arithmetic means ± SEM. Experiments were carried out in triplicate. Data were evaluated with Graph Pad Prism® Version 2.01 software (San Diego, CA, USA). The statistical significance of differences between groups was evaluated by one-way anova, using Tukey's multiple comparisons test as post hoc test. P-values of <0.05 were considered statistically significant. In the experiments involving densitometry, the figures shown are representative of at least three different experiments performed on different days.
Melatonin and the proteosome inhibitor MG 132 were purchased from Sigma-Aldrich® Co (Dorset, UK) and were always freshly prepared as stock solutions in DMSO (Panreac®, Barcelona, Spain) and diluted to the desired concentration in culture medium. The final concentration of DMSO in the culture medium was ≤1% in all experiments and it did not significantly influence cell responses.
Effect of melatonin on cell viability
Our first aim was to evaluate the effect of melatonin treatment on the viability of murine peritoneal macrophage cells in presence of LPS. We evaluated the effect of melatonin (6.25–100 μM) on the growth of these cells by SRB assay. Our data demonstrated that incubation with melatonin for up to 18h, at concentrations up to 100 μM, had no effect on viability of murine macrophages, as determined by the SRB assay (Figure 1).
Melatonin inhibited nitrite production and suppressed the LPS-induced iNOS overexpression
LPS induces the synthesis and release of NO into murine macrophage cell medium by iNOS protein expression (Xie et al., 1992). Nitrite production, as an indicator of NO synthesis, was substantially induced in cells treated with LPS, in comparison with that in untreated cells. However, melatonin treatment significantly reduced nitrite production (P < 0.001 vs. DMSO control) suggesting a possible down-regulation of iNOS enzyme activity (Figure 2A) that was later confirmed by measuring iNOS protein expression with Western blot. Immunoblotting analysis demonstrated a significant decrease in iNOS protein levels, after incubation for 18 h with melatonin at the concentrations assayed (25, 50 and 100 μM; P < 0.01 vs. DMSO control; Figure 2B).
Melatonin induced down-regulation of COX-2 and mPGES1 overexpression induced by LPS
Subsequently, we investigated the effects of melatonin on enzymes related to PGs in inflammation. COX-2 protein expression was clearly induced by LPS treatment (Figure 3A) However, a significant down-regulation of this pro-inflammatory protein was observed in those cells treated with 50 or 100 μM melatonin (P < 0.05 and P < 0.001, respectively, vs. DMSO control; Figure 3B).
It has been reported that mPGES1, one of the PGE2 synthases, is co-localized and functionally coupled with COX-2 (Murakami et al., 2000). In our murine macrophages, LPS stimulation markedly increased expression of mPGES1 protein (Figure 3A). However, exposure to melatonin before LPS stimulation resulted in a significant inhibition of LPS-induced mPGES1 protein expression, in macrophages treated with the highest concentration of melatonin (P < 0.05 vs. DMSO control; Figure 3B).
Effect of melatonin on LPS-induced activation of MAPKs in murine peritoneal macrophages
The MAPK signalling pathways are involved in the expression of many inflammatory protein genes including iNOS, TNF-α and COX-2. To further explore the molecular mechanisms underlying the anti-inflammatory effects of melatonin, we also determined its role on MAPK activation by Western blot analysis, using phosphospecific MAPK antibodies (Figure 4). Cells were incubated in absence or presence of different concentrations of melatonin before LPS stimulation. LPS induced the appearance of phosphorylated (activated) JNK and p38, whereas melatonin treatment, at all doses assayed, inhibited the activation of p38 (P < 0.01 vs. DMSO control; Figure 4), but not that of JNK.
Effect of melatonin on Nrf2-mediated transcriptional activation and HO1 induction in murine peritoneal macrophages
Nrf2 is a key transcription factor that regulates the cellular antioxidant response. Upon cell stimulation, Nrf2 is translocated from the cytosol to the nucleus, and sequentially binds to a promoter sequence called the antioxidant response, element (ARE), resulting in a cytoprotective response characterized by up-regulation of antioxidant enzymes [quinone oxidoreductase-1 (NQO1), HO1 and SOD] and decreased sensitivity to oxidative stress damage (Jaiswal, 2004). Nrf2 also plays a broader role in modulating acute inflammatory responses (Owuor and Kong, 2002). To identify whether melatonin modulated the Nrf2 signalling pathway, we measured the expression of Nrf2 and HO1 protein, by Western blot. As shown in Figure 5A, LPS induced a significant down-regulation of both Nrf2 and HO1 proteins, compared with untreated macrophages. However, incubation with melatonin (100 μM) caused a marked increase in Nrf2 and HO1 expression (P < 0.001 and P < 0.05, respectively, vs. DMSO control; Figure 5B).
Melatonin inhibited NF–κB-mediated activation of transcription and prevented degradation of IkBα in murine peritoneal macrophages
NF-κB is a pleiotropic mediator, controlling several inducible and tissue-specific genes (Lenardo and Baltimore, 1989) and is one of the key regulators of the cellular responses to oxidative stress in mammalian cells (Helenius et al., 2001). Given the relevance of NF-κB to human diseases and the fact that many drugs interfere with NF-κB signalling, this signalling pathway provides a highly attractive target for anti-inflammatory therapy. The activation step allowing NF-κB to leave the cytoplasm involves the ubiquitination of IκBα by the SCF-β-TrCP ubiquitin ligase complex followed by the rapid degradation of ubiquitinated IκBα by the 26S proteasome (Scheidereit, 2006). We therefore tested the effect of melatonin on IκBα degradation and NF-κB activation in murine peritoneal macrophages. As shown in Figure 6, LPS stimulation increased IκBα degradation, which was consistent with an up-regulation of the translocation of p65 protein to the nucleus. On the contrary, pretreatment with melatonin (25, 50 and 100 μM) caused a significant parallel inhibition of NF–κB-mediated transcriptional activation, preventing IκBα degradation and the nuclear translocation of p65 protein in murine macrophages, after LPS stimulation (P < 0.001 vs. DMSO control).
Effects of melatonin on Nrf2 and HO1 protein expression after inhibition of the NF-κB signalling pathway in murine peritoneal macrophages
Our results revealed that melatonin blocked the activation of the NF-κB pathway, impairing the expression of genes related to the anti-inflammatory phase whereas it enhanced some anti-inflammatory signals. As a result, we addressed the causal relationship between NF-κB inhibition and Nrf2 and HO1 overexpression, mediated by melatonin in our model.
The 20S proteasome, the catalytic core of the 26S proteasome complex, is responsible for the breakdown of short-lived regulatory proteins, including Nrf2 and NF-κB (Dreger et al., 2009). In the present study, macrophages were pretreated with 2 μM Z-Leu-Leu-Leu-al (MG 132), a 26S proteasome inhibitor, for 1 h, incubated in presence or absence of LPS (5 μg·mL−1) for 30 min and then treated with melatonin (12.5, 25, 50 and 100 μM). As expected, the nuclear level of p65 protein did not significantly alter after 18 h LPS stimulation in the presence of MG 132, with or without melatonin, in comparison with untreated cells (Figures 6 and 7). Nevertheless, Nrf2 protein expression was unaltered in presence or absence of MG 132 (Figures 5 and 7). Finally, no changes in the expression of the anti-inflammatory HO1 enzyme were detected in LPS-treated cells after incubation with MG 132 in presence or absence of melatonin when compared with untreated cells (Figures 5 and 7).
LPS-stimulated macrophages disrupt the balance of the intracellular redox state, which leads to oxidative stress characterized by a major shift in the cellular redox balance and usually accompanied by ROS-mediated damage (Kang and Lee, 2012). Stimulation of macrophages induces transcription of the iNOS gene and large amounts of NO are generated. NO acts as an intracellular messenger, which modulates the formation of endogenous ROS including hydrogen peroxide, peroxynitrite and other potential oxidants, that orchestrate the inflammatory response (Li et al., 2012). ROS are capable of eliciting a variety of pathological changes, including the peroxidation of lipids, proteins and DNA. Therefore, modulators of ROS production and ROS-induced signalling pathways, especially in macrophages, could represent potential targets for anti-inflammatory intervention (Kim et al., 2012). In the present study, we found that exposure of peritoneal macrophages to LPS resulted in a significant increase in nitrite levels, as an indicator of NO production, and an up-regulation of iNOS expression. However, melatonin inhibited these effects in a concentration-dependent manner. These findings are in accordance with other studies of murine macrophages (Zhang et al., 2004) and J774 and RAW 264.7 cells, stimulated with bacterial LPS (Mayo et al., 2005; Deng et al., 2006). mPGES1 is an efficient downstream enzyme for the production of PGE2 in macrophages activated by LPS (Lazarus et al., 2002) and is co-localized and functionally coupled with COX-2 (Murakami et al., 2000). COX-2, the inducible isoform of COX, is the key enzyme that catalyses the two sequential steps in the biosynthesis of PGs from arachidonic acid, and plays a critical role in the inflammatory response. A selective inhibitor of mPGES1 would be expected to inhibit PGE2 production induced by inflammation while sparing constitutive PGE2 production (Kudo and Murakami, 2005; Wang et al., 2006). In our study, melatonin treatment before LPS stimulation resulted in a significant down-regulation of both proteins, indicating a potential dual action on both COX-2 and mPGES1 enzymes involved in PGE2 synthesis. These results are consistent with those obtained from other studies where melatonin, at non-cytotoxic concentrations, time and concentration-dependently inhibited the induced protein levels and promoter activities of COX-2 in LPS-activated RAW264.7 cells (Mayo et al., 2005; Deng et al., 2006) or stimulated with fimbriae of Porphyromonas gingivalis (Murakami et al., 2012). Furthermore, our results are in agreement with those of Niranjan et al. (2012). These authors, using LPS-stimulated rat astrocytoma cells (C6), found that melatonin reversed LPS-induced changes in mRNA expression of mPGES1 and phosphorylated p38 MAPK. Similarly, melatonin treatment of C6 cells for 24 h significantly decreased LPS-induced nitrosative and oxidative stress and expressions of COX-2 and iNOS. Of the several transcription factors activated by inflammatory stimuli, the NF-κB signalling pathway plays a key role in mediating inflammation and immune responses, through induction of pro-inflammatory cytokines, chemokines and other proteins. NF-κB, as a dimeric transcription factor composed of p65 (RelA), RelB, c-Rel, NF-κB1 (p50/p105) or NF-κB2 (p52/p100) exists in the cytoplasm as an inactive complex with the inhibitory protein, IkBα. When cells are challenged with pro-inflammatory stimuli, for example LPS, IkBα is phosphorylated and subsequently ubiquitinated, allowing NF-κB to translocate to the nucleus. Consequently, NF-κB binds to kB enhancer elements present in the promoter region of many pro-inflammatory genes such as iNOS and COX-2 (Tak and Firestein, 2001; Lee and Surh, 2012).
Moreover, the MAPKs are a family of serine–threonine kinase enzymes that orchestrate the recruitment of gene transcription, protein biosynthesis, cell cycle control, apoptosis, and differentiation and allow cells to respond to oxidative stress and inflammatory stimuli, from their extracellular environment (Munoz and Ammit, 2010). MAPKs include ERKs-1 and -2, JNKs and p38 MAPKs. JNKs, encoded by three genes (JNKs 1–3) while activated by mitogens, are also vigorously stimulated by a variety of environmental stresses, including genotoxins, ischaemia-reperfusion injury, mechanical shear stress, vasoactive peptides and pro-inflammatory cytokines. The p38 MAPKs encoded by four p38 genes are preferentially activated in situ by environmental stresses and pro-inflammatory cytokines such as TNF-α, IL-6, IL-7 and IL-8 in many cell types (Yong et al., 2009; Kyriakis and Avruch, 2012). In effect, MAPKs have been shown to play important roles in iNOS and COX-2 up-regulation induced by various stimuli in mammalian cells (Guha and Mackman, 2001).
Our data showed that treatment with melatonin before 18 h incubation with LPS, significantly prevented IκBα degradation and blocked p65 translocation into the nuclei. In addition, such pre-treatment attenuated the activation of p38 MAPK but was unable to decrease JNK phosphorylation. These data are partially in agreement with Niranjan et al. (2012) and Joo and Yoo (2009), who used LPS-stimulated rat astrocytoma cells or prostate cancer cells (LNCaP), respectively, and found that melatonin reversed LPS-induced changes in mRNA expression of both phosphorylated p38 and JNK MAPKs. Also, in another model (Esposito et al., 2009), melatonin treatment reduced the activation of p38, JNK and ERK1/2 MAPKs, suggesting that the reduction by melatonin of spinal cord injury in mice could also be related to a inhibition of the MAPK signalling pathways.
Altogether, our data suggest that melatonin inhibits iNOS, COX-2 and mPGES1 protein expression by a common transcriptional mechanism modulating the activation of NF-κB and p38 MAPK cascade signalling pathways, suggesting that both the NF-κB transcription factor and the p38 MAPK could be involved in mediating the anti-inflammatory effects of melatonin in murine LPS-activated macrophages.
Immunocompetent cells with melatonin receptors are target cells for its immunomodulatory function (Carrillo-Vico et al., 2003) cooperating during the onset, progression and resolution of inflammation (Soehnlein and Lindbom, 2010). Large amounts of melatonin are produced by all immunocompetent cells, including macrophages, acting as an intracrine, autocrine, and/or paracrine mediator. Recently, it has been suggested that during inflammatory responses, NF-κB induced endogenous synthesis of melatonin in a physiological range, i.e., in pg amounts, in RAW 264.7 macrophages by inducing the transcription of the key enzyme involved in melatonin synthesis arylalkylamine-N-acetyltransferase (AA-NAT) and that macrophage-synthesized melatonin modulated the function of these professional phagocytes in an autocrine manner (Muxel et al., 2009). Our results suggest that treatment with exogenous melatonin, in a pharmacological range, i.e., μg amounts, may modulate NF-κB translocation via AA-NAT, through a negative feedback mechanism contributing to macrophage homeostasis during resolution of inflammation. Nevertheless, further investigations are necessary to substantiate prove this proposal .
Recent reports revealed that melatonin treatment caused a significant up-regulation of LPS-induced Nrf2 and HO1 protein levels. Nrf2 is a key orchestrator of the induction of several antioxidants, which regulates the cellular antioxidant response against ROS. Nrf2 belongs to the ‘cap'n'collar’ basic leucin zipper family of proteins. Under basal conditions, Nrf2 is sequestered in the cytoplasm by its inhibitor Keap1, then ubiquitinylated, and finally degraded by the proteasome. In the presence of oxidative stress, Keap1 releases Nrf2, which can migrate to the nucleus, bind to the antioxidant response element sequence, and induce phase II gene transcription resulting in a cytoprotective response characterized by up-regulation of antioxidant enzymes such as NADPH NQO1, SOD, GSH peroxidase and HO1, and decreased sensitivity to oxidative stress damage (Owuor and Kong, 2002). Also, it has been reported that Nrf2 plays a broader role in modulating acute inflammatory responses (Jung et al., 2010b). On the other hand, HO1 is the inducible isoform of the rate-limiting enzyme of haem degradation. HO regulates the cellular content of the pro-oxidant haem and produces catabolites with physiological functions. HO1 is strongly induced by its substrate haem and by numerous stress stimuli such as UV light, heavy metals, heat shock and hyperoxia. More recently, HO1 has also been recognized to exhibit important immunomodulatory and anti-inflammatory functions (Paine et al., 2010). Our results, in LPS-stimulated macrophages, showed that melatonin increased expression of Nrf2 and the antioxidant HO1 enzyme, in parallel with the decrease of inflammatory mediators such as iNOS, COX-2 and mPGES1, suggesting that melatonin may play a role as an antioxidant defense via the Nrf2/HO1 pathway. Similar results have been obtained by other authors in in vivo experimental models such as dimethylnitrosamine-induced liver injury (Jung et al., 2010a), cisplatin-induced nephrotoxicity (Kilic et al., 2013), in hepatic ischaemia-reperfusion injury (Kang and Lee, 2012), in experimental diabetic neuropathy (Negi et al., 2011) and in interstitial cystitis (Zhang et al.,2013). On the other hand, NF-κB appears to be directly involved in the induction of HO1 gene expression. Increased expression of the HO1 gene is considered to be an adaptive cellular response to survive exposure to environmental stresses (Paine et al.,2010). In order to clarify the role of NF-κB and Nrf2 on HO1 melatonin-mediated overexpression in LPS-stimulated macrophages, we used an inhibitor of NF-κB translocation, MG 132.
As expected, the nuclear p65 protein expression did not significantly alter after 18 h LPS stimulation in the presence of MG 132, with or without melatonin, in comparison with untreated cells, whereas Nrf2 protein expression was maintained unaltered in the presence or absence of MG 132, suggesting that Nrf2 overexpression mediated by melatonin was through a mechanism independent of the NF-κB signalling pathway. Finally, no changes in the expression of the anti-inflammatory HO1 enzyme were detected in LPS-treated cells after incubation with MG 132 in presence or absence of melatonin compared with untreated cells. These results suggest that HO1 melatonin-mediated overexpression could be controlled, at least in part, by NF-κB signalling pathways contributing to the anti-inflammatory effects of melatonin. This relationship has been previously described in human renal proximal tubule cells treated with curcumin and co-incubated with an inhibitor of IKBα phosphorylation, where HO1 induction by curcumin was mediated, at least in part, via transcriptional mechanisms and involved the NF-κB signalling pathway (Hill-Kapturczak et al.,2001). Our results are also in accordance with those from Naidu et al., who found that HO1 gene expression was not up-regulated in phorbol myrisate acetate-activated monocytes from mice, deficient for the NF-κB subunit p65 (Naidu et al., 2008). Similarly, Li et al., showed that HO1 up-regulation was mediated by iNOS and by augmenting NF-κB binding to the region of the HO1 gene promoter in transgenic mice with cardiomyocyte-restricted expression of a dominant negative mutant of IκBα. (Li et al.,2009).
In conclusion, our study showed that melatonin reduced the pro-inflammatory proteins iNOS, COX-2 and mPGES1, and enhanced the expression of HO1 via NF-κB, Nrf2 and p38 MAPK cascade signalling pathways. Thus, melatonin might be a promising target for diseases associated with an overactivation of macrophages.
This work was supported by funds from the Spanish Ministerio de Ciencia e Innovación (AGL 2008-02475, AGL 2011-26949) and Junta de Andalucía (P-10AGR-6609). The authors gratefully acknowledge the assistance of Center for Technology and Innovation Research, University of Seville (CITIUS).