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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.
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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.