ABSTRACT

Melatonin is a neurohormone produced by the pineal gland that regulates sleep and circadian functions. Melatonin also regulates inflammatory and immune processes acting as both an activator and inhibitor of these responses. Melatonin demonstrates endocrine, but also paracrine and autocrine effects in the leukocyte compartment: on one side, leukocytes respond to melatonin in a circadian fashion; on the other side, leukocytes are able to synthesize melatonin by themselves.

With its endocrine and paracrine effects, melatonin differentially modulates pro-inflammatory enzymes, controls production of inflammatory mediators such as cytokines and leukotrienes and regulates the lifespan of leukocytes by interfering with apoptotic processes. Moreover, its potent anti-oxidant ability allows scavenging of oxidative stress in the inflamed tissues. The interesting timing of pro-and anti-inflammatory effects, such as those affecting lipoxygenase activity, suggests that melatonin might promote early phases of inflammation on one hand and contribute to its attenuation on the other hand, in order to avoid complications of chronic inflammation. This review aims at giving a comprehensive overview of the various inflammatory pathways regulated by this pleiotropic hormone.

KEY WORDS

Melatonin; inflammation; oxidative stress; reactive oxygen species; lipoxygenase; NF-B

Introduction

Inflammation is an essential response to tissue injuries induced by physical, chemical or biological insults. Acute and chronic inflammation are essential to restore homeostasis [START_REF] Iwasaki | Regulation of adaptive immunity by the innate immune system[END_REF][START_REF] Cain | Effects of acute and chronic inflammation on B-cell development and differentiation[END_REF]. However, the mechanisms by which activated leukocytes fight germs and tumor cells or eliminate tissue debris in the inflamed area, lead to production of oxidants and/or of cytotoxic cytokines [START_REF] Cain | Effects of acute and chronic inflammation on B-cell development and differentiation[END_REF][START_REF] Davì | Oxidant stress, inflammation and atherogenesis[END_REF].

These processes are finely regulated, but may occasionally escape control and prolong the inflammatory response creating a self-perpetuating condition that produces further damage to the injured tissue. Since chronic inflammation is a major cause of many serious pathological conditions, and is considered by many scientists as a strong tumor promoter [START_REF] Federico | Chronic inflammation and oxidative stress in human carcinogenesis[END_REF]. It is crucial to investigate novel pharmacological approaches that allow to reduce pathologic chronic inflammation without impairing the physiological inflammatory response, since many synthetic anti-inflammatory agents generate considerable side effects.

Pharmaceutical compounds modulating their action in subtle ways (which may be defined as smart compounds), more specifically of natural origins, able to control, regulate and defeat chronic inflammation, are within the focus of nowadays medicinal chemistry research. Endogenous molecules may be exogenously supplemented, thus increasing overall body levels, and changing timing or localization within the body with respect to their endogenous production.

Melatonin is very attractive from this point of view as it may act as a regulator of the inflammatory cell compartment with a potent anti-oxidant potential able to reduce the oxidative environment of chronic inflammation and to regulate leukocyte function and number [START_REF] Carrillo-Vico | Evidence of melatonin synthesis in human lymphocytes and its physiological significance: possible role as intracrine, autocrine and/or paracrine substance[END_REF][START_REF] Tan | Melatonin: a hormone, a tissue factor, an autocoid, a paracoid, and an antioxidant vitamin[END_REF].

We analyze here data describing the possible modulation of inflammatory functions with a focus on the anti-oxidant effects of melatonin on its ability to alter production of pro-inflammatory molecules in vivo and in vitro. Moreover, this review will cover the effects that melatonin exerts on the abundance of leukocytes via alteration of their proliferation and apoptosis.

Melatonin synthesis and its targets

Melatonin (N-acetyl-5-methoxytryptamine) is derived from tryptophan via enzymatic conversion reactions (see Figure 1 for details) and is mainly produced by the pineal gland.

Melatonin plasma concentration varies according to a circadian cycle and this cyclic production determines the periodic effects at a systemic level [START_REF] Reiter | Pineal melatonin: cell biology of its synthesis and of its physiological interactions[END_REF]. Melatonin is evolutionary conserved and exerts many regulatory functions by modulating cellular behavior via binding to specific receptors and intracellular targets (Figure 2) [START_REF] Pandi-Perumal | Physiological effects of melatonin: role of melatonin receptors and signal transduction pathways[END_REF][START_REF] Carlson | Melatonin signal transduction in hamster brain: inhibition of adenylyl cyclase by a pertussis toxin-sensitive G protein[END_REF].

Target cells express specific high affinity receptors, namely MT1/MT2, on plasma membrane [START_REF] Sm | Cloning and characterization of a mammalian melatonin receptor that mediates reproductive and circadian responses[END_REF], able to trigger intracellular signaling by adenylate cyclase or G-proteins (Figure which melatonin stimulates these signal transduction pathways have been extensively studied [START_REF] Hardeland | Melatonin: signaling mechanisms of a pleiotropic agent[END_REF][START_REF] Rodriguez | Regulation of antioxidant enzymes: a significant role for melatonin[END_REF][START_REF] Vanecek | Melatonin inhibits cyclic AMP and cyclic GMP accumulation in the rat pituitary[END_REF][START_REF] Mcarthur | Melatonin action and signal transduction in the rat suprachiasmatic circadian clock: activation of protein kinase C at dusk and dawn[END_REF][START_REF] Vanecek | Melatonin inhibits gonadotropin-releasing hormone-induced elevation of intracellular Ca2+ in neonatal rat pituitary cells[END_REF][START_REF] Vanecek | Melatonin inhibits cyclic AMP and cyclic GMP accumulation in the rat pituitary[END_REF][START_REF] Vanecek | Melatonin modulates diacylglycerol and arachidonic acid metabolism in the anterior pituitary of immature rats[END_REF][START_REF] Rodriguez | Regulation of antioxidant enzymes: a significant role for melatonin[END_REF][START_REF] Chan | Melatonin mt1 and MT2 receptors stimulate c-Jun N-terminal kinase via pertussis toxin-sensitive and -insensitive G proteins[END_REF][START_REF] Pablos | Both melatonin and putative nuclear melatonin receptor agonist CGP 52608 stimulate glutathione peroxidase and glutathione reductase activities in mouse brain in vivo[END_REF], little is known about the resulting cellular effects: MT1/MT2 receptors have been shown to mediate melatonin-dependent decrease of tumor cell proliferation [START_REF] Hill | Molecular mechanisms of melatonin anticancer effects[END_REF] and anti-apoptotic effects [START_REF] Radogna | Melatonin antagonizes apoptosis via receptor interaction in U937 monocytic cells[END_REF][START_REF] Radogna | Neuroprotection by melatonin on astrocytoma cell death[END_REF][START_REF] Das | The inhibition of apoptosis by melatonin in VSC4.1 motoneurons exposed to oxidative stress, glutamate excitotoxicity, or TNF-alpha toxicity involves membrane melatonin receptors[END_REF]; MT1 seems to be responsible for the general response to photoperiodic signals [START_REF] Yasuo | Melatonin transmits photoperiodic signals through the MT1 melatonin receptor[END_REF].

High affinity nuclear receptors to melatonin have also been identified (Figure 2B). They belong to the RZR/ROR nuclear hormone receptor family; melatonin binds with high affinity to RZR-beta [START_REF] Becker-André | Pineal gland hormone melatonin binds and activates an orphan of the nuclear receptor superfamily[END_REF][START_REF] Wiesenberg | Transcriptional activation of the nuclear receptor RZR alpha by the pineal gland hormone melatonin and identification of CGP 52608 as a synthetic ligand[END_REF]. Upon activation, this complex down-regulates the 5-lipoxygenase (LOX) whose promoter possesses an RZR-beta-repressor binding site [START_REF] Carlberg | The orphan receptor family RZR/ROR, melatonin and 5lipoxygenase: an unexpected relationship[END_REF]; subsequently, 5-LOX expression is reduced by melatonin in cells bearing this RZR-beta receptor [START_REF] Steinhilber | The nuclear receptor for melatonin repress 5-lipoxygenase gene expression in human B lymphocytes[END_REF].

Cells expressing either one of those receptors are targets of the hormonal effect of melatonin and respond to its nocturnal low nanomolar plasma levels. Additional binding to intracellular targets in the micromolar range, such as the enzymes hydroquinone [START_REF] Boutin | Melatonin binding site MT3 is QR2: state of the art[END_REF] and calmodulin [START_REF] Benitez-King | Binding of 3H-melatonin to calmodulin[END_REF] (Figure 2C) have been reported. These interactions trigger signaling functions including microtubule regulation [START_REF] Benítez-King | Calmodulin mediates melatonin cytoskeletal effects[END_REF] and possibly phospholipase A2 activation [START_REF] Radogna | Lipoxygenase-mediated pro-radical effect of melatonin via stimulation of arachidonic acid metabolism[END_REF]; since hydroquinone and calmodulin are ubiquitous, most cells are targets of melatonin; however, low affinity allows productive interaction only at concentrations much higher than those produced by the pineal gland, suggesting that other mechanisms of melatonin production or accumulation may take place.

In addition to the pineal gland, which is the major source of melatonin, other tissues like retina [START_REF] Faillace | Evidence for local synthesis of melatonin in golden hamster retina[END_REF], gastrointestinal tract [START_REF] Huether | Effect of tryptophan administration on circulating melatonin levels in checks and rats: evidence for stimulation of melatonin synthesis and release in the gastrointestinal tract[END_REF][START_REF] Bubenik | Gastrointestinal melatonin: localization, function and clinical relevance[END_REF] , skin [START_REF] Pisarchik | Serotoninergic and melatoninergic systems are fully expressed in human skin[END_REF] and leukocytes [START_REF] Carrillo-Vico | Evidence of melatonin synthesis in human lymphocytes and its physiological significance: possible role as intracrine, autocrine and/or paracrine substance[END_REF] (both in peripheral blood [START_REF] Lopez-Gonzalez | Characterization of melatonin binding sites in human peripheral blood neutrophils[END_REF] and in bone marrow [START_REF] Conti | Evidence for melatonin synthesis in mouse and human bone marrow cells[END_REF][START_REF] Tan | Identification of highly elevated levels of melatonin in bone marrow: its origin and significance[END_REF]) synthesize melatonin. Melatonin produced by these nonendocrine organs is not regulated by circadian cycles but rather respond to other signals, exerting in fact a paracrine or autocrine effect [START_REF] Carrillo-Vico | Evidence of melatonin synthesis in human lymphocytes and its physiological significance: possible role as intracrine, autocrine and/or paracrine substance[END_REF]. Local melatonin levels may differ from those regulated by its neuro-endocrine functions; indeed, melatonin reaches the highest body concentration in the bone marrow (micromolar concentrations), exceeding plasma levels of at least two orders of magnitude [START_REF] Tan | Identification of highly elevated levels of melatonin in bone marrow: its origin and significance[END_REF]. This accumulation may result from the intrinsic melatonin synthesis machinery of the leukocyte.

As a matter of fact, melatonin finely regulates leukocyte function and number and contributes to the control of inflammatory tissue [START_REF] Carrillo-Vico | A review of the multiple actions of melatonin on the immune system[END_REF]. The effects of melatonin on leukocytes imply paracrine or autocrine responses that superimpose on the neuro-endocrine hormone response. In vivo studies with pinealectomized animals [START_REF] Gobbo | Pinealectomy inhibits interleukin-2 production and natural killer activity in mice[END_REF] allow to discriminate between hormonal and paracrine responses, whereas the study of melatonin synthesis and functions in cultured non-pineal cells [START_REF] Carrillo-Vico | Evidence of melatonin synthesis in human lymphocytes and its physiological significance: possible role as intracrine, autocrine and/or paracrine substance[END_REF] provides information of the autocrine effects of melatonin.

Neuro-endocrine melatonin: systemic effects of pineal melatonin on immune system

It is an established notion that the nervous and endocrine systems can interact with the immune system in order to modulate its function [START_REF] Blalock | The syntax of immune-neuroendocrine communication[END_REF]; indeed, many neurotransmitters, neuroendocrine factors and hormones can alter immune function [START_REF] Ader | Psychoneuroimmunology: interactions between the nervous system and the immune system[END_REF]. Also melatonin is able to modulate immune functions through its neuro-endocrine action. Melatonin acts as a regulator of circadian rhythms [START_REF] Malpaux | Biology of mammalian photoperiodism and the critical role of the pineal gland and melatonin[END_REF] in a hormone-like fashion by affecting target cells and by modulating other functions depending on the photoperiod including regulation of photoperiodic oscillations of the immune/inflammatory response [START_REF] Scheff | Modeling the influence of circadian rhythms on the acute inflammatory response[END_REF][START_REF] Malpaux | Biology of mammalian photoperiodism and the critical role of the pineal gland and melatonin[END_REF]. Leukocytes possess melatonin specific receptors including both MT1/MT2 and nuclear RZR, and this provides the molecular basis for the sensitivity of leukocytes to melatonin.

A strong indication of the effect of melatonin produced by the pineal gland on inflammatory cells via its endocrine activity was obtained in the Gerbil and in the Syrian Hamster. Here the artificial shortening of day lengths or alternatively melatonin injections induce an increase in thymus weight [START_REF] Vaughan | Influence of melatonin constant light, or blinding on reproductive system of gerbils (Meriones unguiculatus)[END_REF] and spleen hypertrophy [START_REF] Vaughan | Splenic hypertrophy and extramedullary hematopoiesis induced in male Syrian hamster by short photoperiod or melatonin injection and reversed by melatonin pellets or pinealectomy[END_REF], implying a melatonin-mediated photoperiodic effect on the immune system. Furthermore, it was demonstrated that melatonin controls diurnal and seasonal rhythms of leukocyte proliferation [START_REF] Drazen | Melatonin enhancement of splenocyte proliferation is attenuated by luzindole, a melatonin receptor antagonist[END_REF], of cytokine production [START_REF] Gobbo | Pinealectomy inhibits interleukin-2 production and natural killer activity in mice[END_REF][START_REF] Scheff | Modeling the influence of circadian rhythms on the acute inflammatory response[END_REF] and NK cell activity [START_REF] Matsumoto | Total sleep deprivation induces an acute and transient increase in NK cell activity in healthy young volunteers[END_REF] in mammalian bone marrow cells. In other studies, the nocturnal peak of melatonin was associated with the proliferation peak of granulocytes and macrophage progenitor cells in mice [START_REF] Haldar | Effect of the pineal gland on circadian rhythmicity of colony forming units for granulocytes and macrophages (CFU-GM) from rat bone marrow cell cultures[END_REF].

Data from the studies of the effects of experimental pinealectomy further contribute to a better understanding of the underlying mechanisms. It was described that the removal of the pineal gland causes partial (and transient) impairment of immune response in rats [START_REF] Gobbo | Pinealectomy inhibits interleukin-2 production and natural killer activity in mice[END_REF][START_REF] Haldar | Effect of the pineal gland on circadian rhythmicity of colony forming units for granulocytes and macrophages (CFU-GM) from rat bone marrow cell cultures[END_REF][START_REF] Martins E Jr | Melatonin modulates allergic lung inflammation[END_REF]; moreover, NK activity and IL-2 production were described to be reduced in rats after pinealectomy [START_REF] Gobbo | Pinealectomy inhibits interleukin-2 production and natural killer activity in mice[END_REF].

Interestingly, melatonin administration in pinealectomized animals reverses the adverse effects on immune response. For example, Martins et al., in an experimental model of allergic airway inflammation, showed that melatonin administration to pinealectomized rats restores the ability of cells to migrate from the bone marrow to the broncho-alveolar fluid, which was prevented after pineal gland removal, implying that neuro-endocrine melatonin is important in the control of cell recruitment from the bone marrow and the migration of those cells to the lung [START_REF] Martins E Jr | Melatonin modulates allergic lung inflammation[END_REF] As the melatonin-dependent effects of pinealectomy on the immune response appear to be partial, this further indicates that other compartments can overcome or contribute to immunomodulatory effects triggered by melatonin. Accordingly melatonin may act as a paracrine or autocrine regulator in leukocyte communication independent of the pineal gland. Indeed Jurkat T cells produce interleukine-2 (IL-2) as a result of endogenous melatonin signaling and Lardone et al. demonstrated that IL-2 production is hampered by specifically inhibiting melatonin signaling by the MT1/MT2 antagonist luzindole [START_REF] Lardone | A novel interplay between membrane and nuclear melatonin receptors in human lymphocytes: significance in IL-2 production[END_REF]. Recently it was discovered that in bone marrow, melatonin reaches micromolar concentrations, that is much higher than those found in the blood stream, suggesting that the paracrine/autocrine immunoregulatory role of melatonin might be also mediated by binding to the low affinity targets; a putative signal transduction pathway triggered by melatonin binding to calmodulin (affinity = 63 µM [START_REF] Romero | Membrane-bound in Xenopus laevis oocytes as a novel binding site for melatonin[END_REF]) leads to transient activation of phospholipase A2 and lipoxygenase activation [START_REF] Radogna | Lipoxygenase-mediated pro-radical effect of melatonin via stimulation of arachidonic acid metabolism[END_REF] (Figure 3).

Paracrine effects of melatonin on the white blood cell compartment

Effect of exogenous melatonin on inflammatory parameters

The potential use of melatonin as a pharmaceutical agent derives from numerous in vitro studies where melatonin doses exceeding the nocturnal plasma levels are required to exert clear effects. At supra-physiological concentrations, melatonin induces T-cell proliferation and upregulation of pro-inflammatory cytokines [START_REF] Carrillo-Vico | A review of the multiple actions of melatonin on the immune system[END_REF][START_REF] Currier | Exogenous melatonin: quantitative enhancement in vivo of cells mediating non-specific immunity[END_REF] . Exogenous melatonin administration increases the proliferative response of rat lymphocytes [START_REF] Pahlavani | In vitro effects of melatonin on mitogen-induced lymphocyte proliferation and cytokine expression in young and old rats[END_REF] , increases the number of NK cells [START_REF] Angeli | Effect of exogenous melatonin on human natural killer (NK) cell activity. An approach to the immunomodulatory role of the pineal gland[END_REF][START_REF] Huang | Effect of melatonin and electroacupuncture (EA) on NK cell activity, interleukin-2 production and POMC-derived peptides in traumatic rats[END_REF], stimulates the pro-inflammatory cytokines IL-1 and TNF- [START_REF] Shafer | Assessment of melatonin's ability to regulate cytokine production by macrophage and microglia cell types[END_REF][START_REF] Pioli | Melatonin increases antigen presentation and amplifies specific and non specific signals for T-cell proliferation[END_REF] and enhances phagocytosis [START_REF] Sanchez | Tryptophan administration in rats enhances phagocytic function and reduces oxidative metabolism[END_REF]. Melatonin exerts a concentration-dependent effect on the immune system. Indeed, increasing concentrations of melatonin induce T-cell proliferation in a dose-dependent way. In addition, it was demonstrated that pharmacological doses of melatonin inhibit INF- production in a range of 0.1 to 1mM [START_REF] Arzt | Immunomodulation by indoleamines: serotonin and melatonin action on DNA and interferongamma synthesis by human peripheral blood mononuclear cells[END_REF]. In some systems, the modulation of apoptosis requires high melatonin doses [START_REF] Radogna | Melatonin antagonizes apoptosis via receptor interaction in U937 monocytic cells[END_REF][START_REF] Radogna | Neuroprotection by melatonin on astrocytoma cell death[END_REF]. Whether or not these effects have a physiological significance, occurring as a paracrine or autocrine response, i.e., in microenvironments with an elevated melatonin concentration like in the bone marrow, is presently investigated. In any case, these findings designate melatonin as a potential exogenous pharmacological modulator of the inflammatory response.

Melatonin, ROS and inflammation

Oxidative stress can be defined as the imbalance between cellular oxidant species like reactive oxygen species (ROS) production and antioxidant potential. ROS are involved in a variety of different cellular processes ranging from physiological to pathological responses. It is well known that ROS can promote cell survival, proliferation and differentiation at physiological levels, but also cell death by apoptosis or necrosis at higher levels [START_REF] Yao | Oxidative stress and chromium(VI) carcinogenesis[END_REF]. Oxidative stress has a direct toxic A c c e p t e d M a n u s c r i p t -7 -effect on cells, which leads to lipid peroxidation, protein oxidation or DNA damage; it plays a causative or adjuvant role in almost all human pathologies, including cancer and neurodegeneration, and is involved in aging and chronic inflammatory pathologies. To cope with oxidative stress, a battery of enzymes able to scavenge ROS are up-regulated by intracellular signaling [START_REF] Dröge | Free radicals in the physiological control of cell function[END_REF].

Oxidative phenomena play a key role in signaling and management of the initial phase of inflammation. Activated neutrophils generate an oxidative burst that directs toxicity towards invading microbes [START_REF] Bogdan | Reactive oxygen and reactive nitrogen intermediates in innate and specific immunity[END_REF]; ROS generation in the damaged tissue produce a concentration gradient that directs leukocyte recruitment to the site of tissue injury [START_REF] Niethammer | A tissue-scale gradient of hydrogen peroxide mediates rapid wound detection in zebrafish[END_REF]. Moreover, ROS induce monocyte maturation [START_REF] Cristofanon | Oxidationdependent maturation and survival of explanted blood monocytes via Bcl-2 up-regulation[END_REF] and promote adhesion to endothelial cells allowing diapedesis. Thus, beyond its anti-septic action, ROS generation can regulate leukocyte recruitment and maturation.

ROS generation also activates the intracellular inflammatory signaling pathways leading to the release of the inflammatory mediators, including cytokines produced by the activation of Redox-regulated transcription factors such as NF-B [START_REF] Kim | TNF-alpha-induced upregulation of intercellular adhesion molecule-1 is regulated by a Rac-ROS-dependent cascade in human airway epithelial cells[END_REF][START_REF] Cristofanon | Oxidative, multistep activation of the noncanonical NF-kappaB pathway via disulfide Bcl-3/p50 complex[END_REF]. Thus, ROS can promote and drive the inflammatory response in various tissues and can stimulate the signaling pathways triggered by inflammatory conditions [START_REF] Martinon | Signaling by ROS drives inflammasome activation[END_REF].

If tissue healing is delayed or fails, inflammation may become chronic, increase tissue damage and induce the recruitment of additional leukocytes, producing a vicious cycle and leading to organ failure or eventually to cancer. This is characterized by chronic ROS production. An efficient anti-inflammatory therapeutic strategy requires anti-oxidants to stop this vicious cycle and to reduce the burden of inflammatory mediators at the inflammatory site.

Melatonin is a powerful anti-oxidant [START_REF] Tan | Melatonin: A potent, endogenous hydroxyl radical scavenger[END_REF] as it has been shown to scavenge different types of free radicals in vitro [START_REF] Poeggeler | Melatonin and structurally related endogenous indoles act as potent electron donors and radical scavengers in vitro[END_REF], in body fluids [START_REF] Plessis | The in vitro effects of melatonin on human sperm function and its scavenging activities on NO and ROS[END_REF] and in cells [START_REF] Zavodnik | Melatonin directly scavenges free radicals generated in red blood cells and a cell-free system:chemiluminescence measurements and theoretical calculations[END_REF]. Furthermore, melatonin plays an important role in activating anti-oxidant defenses such as superoxide dismutase (SOD), catalase (CAT), glutathione peroxidase (GSH-Px), glutathione reductase (GSH-Rd) and glucose-6phosphate dehydrogenase (G6PD) [START_REF] Bharti | Pineal proteins upregulate specific antioxidant defense systems in the brain[END_REF][START_REF] Hardeland | Neuroprotection by radical avoidance: search for suitable agents[END_REF].

Both effects allow melatonin to reduce the extent of ROS, improving oxidative related pathologies such as hypertension [START_REF] Reiter | The circadian melatonin rhythm and its modulation: possible impact on hypertension[END_REF], atherosclerosis [START_REF] Broncel | Melatonin in the treatment of atherosclerosis[END_REF], cancer [START_REF] Ravindra | Melatonin in pathogenesis and therapy of cancer[END_REF], ischemia [START_REF] Reiter | When melatonin gets on your nerves: its beneficial actions in experimental models of stroke[END_REF] or neurodegenerative diseases [START_REF] Skaper | Excitotoxicity, oxidative stress, and the neuroprotective potential of melatonin[END_REF] . Moreover it was suggested that melatonin prevents aging [START_REF] Reiter | Reactive oxygen intermediates, molecular damage, and aging. Relation to melatonin[END_REF].

Effects of melatonin on the arachidonic acid metabolism

The inflammatory response starts with an insult that activates and recruits leukocytes to the inflamed tissue through recruitment of different molecular mediators; an important role in the early phase is played by phospholipase A2 (PLA2), which cleaves membrane phospholipids liberating membrane bound arachidonic acid (AA); arachidonic acid is then processed by cyclooxygenases A c c e p t e d M a n u s c r i p t -8 -(COX) and LOX to produce key inflammatory mediators such as prostaglandins and leukotrienes [START_REF] González-Périz | New approaches to the modulation of the cyclooxygenase-2 and 5lipoxygenase pathways[END_REF], respectively. After experimental inflammation induction in vivo and ex vivo, melatonin was shown to prevent or reduce the inflammatory-derived activation of PLA2 [START_REF] Li | Negative regulation of cytosolic phospholipase A(2) by melatonin in the rat pineal gland[END_REF] , LOX [START_REF] Steinhilber | The nuclear receptor for melatonin repress 5lipoxygenase gene expression in human B lymphocytes[END_REF] and COX [START_REF] Deng | Melatonin suppresses macrophage cyclooxygenase-2 and inducible nitric oxide synthase expression by inhibiting p52 acetylation and binding[END_REF]. The reduction of PLA2 [START_REF] Li | Negative regulation of cytosolic phospholipase A(2) by melatonin in the rat pineal gland[END_REF] and LOX [START_REF] Steinhilber | The nuclear receptor for melatonin repress 5lipoxygenase gene expression in human B lymphocytes[END_REF] up-regulation by melatonin occurs via nonoxidative mechanisms, implying the engagement of MT1/MT2 [START_REF] Li | Negative regulation of cytosolic phospholipase A(2) by melatonin in the rat pineal gland[END_REF] or ROR/RZR [START_REF] Steinhilber | The nuclear receptor for melatonin repress 5lipoxygenase gene expression in human B lymphocytes[END_REF] receptors; thus, many different actions of melatonin including receptor stimulation or radical scavenging seem to cooperate to achieve the same final goal, that is to interfere with the inflammatory cascade.

Moreover, recent evidence shows that melatonin activates PLA2 and 5-LOX [START_REF] Radogna | Lipoxygenase-mediated pro-radical effect of melatonin via stimulation of arachidonic acid metabolism[END_REF] as a consequence to its binding to calmodulin [START_REF] Radogna | Lipoxygenase-mediated pro-radical effect of melatonin via stimulation of arachidonic acid metabolism[END_REF][START_REF] Jenkins | Identification of the calmodulin-binding domain of recombinant calcium-independent phospholipase A2beta. Implications for structure and function[END_REF], leading to mobilization of arachidonic acid (AA) from phospholipids. Arachidonic acid then activates 5-LOX triggering production of endogenous monohydroxy-eicosatetraenoic acids (HETEs) [START_REF] Radogna | Lipoxygenase-mediated pro-radical effect of melatonin via stimulation of arachidonic acid metabolism[END_REF]. Interestingly, the down-regulation of 5-LOX (and PLA2) requires the engagement of RZR (or MT1/MT2), receptors [START_REF] Li | Negative regulation of cytosolic phospholipase A(2) by melatonin in the rat pineal gland[END_REF][START_REF] Steinhilber | The nuclear receptor for melatonin repress 5lipoxygenase gene expression in human B lymphocytes[END_REF] but its activation instead requires melatonin to bind to calmodulin (Figure 3). We can conclude that the stimulation by melatonin of different targets produces different effects, altogether suggesting a sophisticated regulation.

The activation of 5-LOX and PLA2 activity by melatonin is a transient phenomenon and extinguishes within 2-3 hrs, possibly because of the down-regulation of either 5-LOX or PLA2.

These observations are consistent with the view that melatonin favors the early phases of inflammation on one side, in fact, LOX induces the production of leukotriene B, which promotes diapedesis by vessel permeabilization, and acts as a chemo-attractant [START_REF] Pidgeon | Lipoxygenase metabolism: roles in tumor progression and survival[END_REF][START_REF] Islam | The leukotriene B4 lipid chemoattractant receptor BLT1 defines antigen-primed T cells in humans[END_REF]. On the other side, melatonin reduces the inflammatory process when it proceeds towards the chronic phase.

Melatonin also inhibits the activity of the enzyme nitric oxide synthase (NOS) [START_REF] Bettahi | Physiological concentrations of melatonin inhibit nitric oxide synthase activity in the rat hypothalamus[END_REF][START_REF] Guerrero | Melatonin prevents increases in neural nitric oxide and cycle GMP production after transient brain ischemia and reperfusion in the Mongolian gerbil (Meriones unguiculatus)[END_REF][START_REF] Pozo | Inhibition of cerebellar nitric oxide synthase and cyclic GMP production by melatonin via complex formation with calmodulin[END_REF], which catalyzes the formation of NO•. In inflamed tissues, NO released after cytokine stimulation contributes to the development of micro-vessel damage and vascular hyporeactivity [START_REF] Tesse | Upregulation of proinflammatory proteins through NF-kappaB pathway by shed membrane microparticles results in vascular hyporeactivity[END_REF]. Thus, melatonin-related down-regulation of NOS could contribute to melatonin"s prevention of inflammation.

It was reported by that melatonin inhibits iNOS expression via down-regulation of NF-B [START_REF] Alonso | Melatonin inhibits the expression of the inducible isoform of nitric oxide synthase and nuclear factor kappa B activation in rat skeletal muscle[END_REF] on one hand but is also able to activate NF-B, thus regulating the expression of adhesion molecules on circulating leukocytes on the other hand [START_REF] Cristofanon | Intracellular prooxidant activity of melatonin induces a survival pathway involving NF-kappaB activation[END_REF][START_REF] Cecon | Daily variation of constitutively activated nuclear factor kappa B (NFKB) in rat pineal gland[END_REF]. Thus, melatonin plays a multifaceted role depending largely on the targets involved.

Role of melatonin in leukocyte survival or apoptosis

Leukocytes have to perform briefly but efficiently in an oxidative environment that may cause mutations or cell death. It is nevertheless important that cells warrant a prolonged anti- Apoptosis plays an important role in inflammatory responses but also in the regulation of the maturation rate of B and T cells [START_REF] De | The apoptotic pathway as a therapeutic target in sepsis[END_REF][START_REF] Bolitho | Apoptosis induced by the lymphocyte effector molecule perforin[END_REF]. In particular, melatonin was shown to regulate the apoptosis of B and T cells [START_REF] Pedrosa | Campa A Melatonin protects CD4+ T cells from activation-induced cell death by blocking NFAT-mediated CD95 ligand upregulation[END_REF]104]. Indeed, melatonin inhibits apoptosis of precursor B cells in mouse bone marrow [START_REF] Yu | Melatonin inhibits apoptosis during early B-cell development in mouse bone marrow[END_REF]. Moreover, an anti-apoptotic effect on different leukocytes was also demonstrated [START_REF] Radogna | Melatonin antagonizes apoptosis via receptor interaction in U937 monocytic cells[END_REF][START_REF] Bolitho | Apoptosis induced by the lymphocyte effector molecule perforin[END_REF].

The mechanisms of the anti-apoptotic action of melatonin in leukocytes have been investigated. Studies in peripheral blood have suggested that melatonin inhibits apoptotic processes via its antioxidant properties [START_REF] Sainz | Melatonin and cell death: differential actions on apoptosis in normal and cancer cells[END_REF], which is in line with many evidences showing that in some instances the apoptotic process occurs via oxidative signaling [START_REF] Ortona | Redox state, cell death and autoimmune diseases: a gender perspective[END_REF]. Alternative mechanisms have been proposed; in U937 monocytes, melatonin reduces apoptosis via intracellular signal transduction stimulated by interaction with MT1/MT2 plasma membrane receptors [START_REF] Radogna | Melatonin antagonizes apoptosis via receptor interaction in U937 monocytic cells[END_REF]. However, the majority of reports indicate that the anti-apoptotic of melatonin occurs by acting on the abundance of the two main members of the Bcl-2 family, namely the pro-apoptotic Bax and the anti-apoptotic Bcl-2 proteins [START_REF] Radogna | Melatonin antagonizes the intrinsic pathway of apoptosis via mitochondrial targeting of Bcl-2[END_REF].

Bcl-2 exerts its well-known anti-apoptotic effect by inhibiting the action of Bax [START_REF] Lucken-Ardjomande | Regulation of Bcl-2 proteins and of the permeability of the outer mitochondrial membrane[END_REF], which induces apoptosis by forming pores in the outer mitochondrial membrane, permeabilizing it to the passage of pro-apoptotic factors such as cytochrome c [START_REF] Lucken-Ardjomande | Regulation of Bcl-2 proteins and of the permeability of the outer mitochondrial membrane[END_REF]. The balance between Bax and Bcl-2 determines the propensity of cells to respond to a given insult by apoptosis or survival [START_REF] Lucken-Ardjomande | Regulation of Bcl-2 proteins and of the permeability of the outer mitochondrial membrane[END_REF].

Melatonin is able to shift the balance towards a cell-protective state [START_REF] Radogna | Melatonin antagonizes the intrinsic pathway of apoptosis via mitochondrial targeting of Bcl-2[END_REF]. Indeed, many studies have demonstrated that its anti-apoptotic effect involves the down-regulation of Bax [START_REF] Maity | Melatonin reduces indoethacininduced gastric mucosal cell apoptosis by preventing mitochondrial oxidative stress and the activation of mitochondrial pathway of apoptosis[END_REF][START_REF] Chetsawang | Melatonin protects against hydrogen peroxide-induced cell death signaling in SH-SY5Y cultured cells: involvement of nuclear factor kappa B, Bax and Bcl-2[END_REF][START_REF] Juknat | Melatonin prevents hydrogen peroxideinduced Bax expression in cultured rat astrocytes[END_REF][START_REF] Espino | Melatonin reduces apoptosis induced by calcium signaling in human leukocytes: Evidence for the involvement of mitochondria and Bax activation[END_REF] and /or the up-regulation of Bcl-2 [START_REF] Maity | Melatonin reduces indoethacininduced gastric mucosal cell apoptosis by preventing mitochondrial oxidative stress and the activation of mitochondrial pathway of apoptosis[END_REF][START_REF] Ling | Protective effect of melatonin on injuried cerebral neurons is associated with bcl-2 protein over-expression[END_REF][START_REF] Cristofanon | Intracellular prooxidant activity of melatonin induces a survival pathway involving NF-kappaB activation[END_REF] that occurs via activation of NF-B [START_REF] Cristofanon | Intracellular prooxidant activity of melatonin induces a survival pathway involving NF-kappaB activation[END_REF]. Besides altering their abundance, melatonin could alter Bax and Bcl-2 activity in other ways. Oxidative alterations cause Bax disulfide dimerization, and the resulting conformational changes are sufficient for its translocation to mitochondria independently of apoptosis [START_REF] D'alessio | Oxidative Bax dimerization promotes its translocation to mitochondria independently of apoptosis[END_REF]. However, melatonin maintains Bax in a monomeric state within mitochondria [START_REF] Radogna | Melatonin antagonizes the intrinsic pathway of apoptosis via mitochondrial targeting of Bcl-2[END_REF]. The inhibition of Bax activation may be due to melatonin's anti-oxidant ability, and/or to the simultaneous re-localization of Bcl-2 [START_REF] Radogna | Melatonin antagonizes the intrinsic pathway of apoptosis via mitochondrial targeting of Bcl-2[END_REF] to mitochondria, thus co-localizing with (and sequestering) Bax. Indeed, the persistence of Bax monomers in cells induced to apoptosis in the presence of melatonin results from the prevalence of a Bax-Bcl-2 heterodimer over the active Bax dimers within mitochondria, thus, blocking both cytochrome C release and apoptosis.

A c c e p t e d M a n u s c r i p t -10 -It is interesting to note that melatonin has developed various independent strategies to inhibit the intrinsic pathway of apoptosis at the level of Bax activation, inducing a pro-survival pathway and by this way maintaining viability of cells at the inflammatory site.

Role of melatonin in maintaining and restoring human health

Melatonin controls the onset and progression of many human pathological states by multiple mechanisms asdescribed by studies reporting effects of clinical trials.

Alterations of circadian rhythms implying variations of the 24-h light/dark cycle, play a critical role in the development and progression of many diseases. Several mediators of the inflammatory response, including cytokines and hormones, have been observed to undergo significant diurnal plasma concentration variations [START_REF] Scheff | Modeling the influence of circadian rhythms on the acute inflammatory response[END_REF][START_REF] Blalock | The syntax of immune-neuroendocrine communication[END_REF][START_REF] Ader | Psychoneuroimmunology: interactions between the nervous system and the immune system[END_REF][START_REF] Malpaux | Biology of mammalian photoperiodism and the critical role of the pineal gland and melatonin[END_REF]. Indeed, a mathematical model validated by experimental data and clinical observations, demonstrates the interplay between inflammation and circadian rhythms [START_REF] Scheff | Modeling the influence of circadian rhythms on the acute inflammatory response[END_REF]. Melatonin is synthesized in the suprachiasmatic nucleus according to a circadian rhythm, thus causing diurnal oscillation of its blood levels. This regulation allows melatonin to coordinate circadian rhythms, including the sleep-wake cycle, thus possessing a beneficial and therapeutic effect as demonstrated by the reports of clinical trials with administration of melatonin [START_REF] Boeve | Melatonin for treatment of REM sleep behavior disorder in neurologic disorders: results in 14 patients[END_REF].

Melatonin is widely used to treat various sleep disorders [START_REF] Dahlitz | Delayed sleep phase syndrome response to melatonin[END_REF][START_REF] Mundey | Phase-dependent treatment of delayed sleep phase syndrome with melatonin[END_REF][START_REF] Oldani | Melatonin and delayed sleep phase syndrome: Ambulatory polygraphic evaluation[END_REF][START_REF] Lapierre | Melatonin treatment of a non-24-hour sleep-wake cycle in a blind retarded child[END_REF][START_REF] Folkard | Melatonin stabilises sleep onset time in a blind man without entrainment of cortisol or temperature rhythms[END_REF][START_REF] Arendt | Synchronisation of a disturbed sleep-wake cycle in a blind man by melatonin treatment (letter)[END_REF][START_REF] Sack | Melatonin administration to blind people: Phase advances and entrainment[END_REF][START_REF] Palm | Correction of non-24-hour sleep/wake cycle by melatonin in a blind retarded boy[END_REF][START_REF] Jan | The treatment of sleep disorders with melatonin[END_REF][START_REF] Jan | Use of melatonin in the treatment of paediatric sleep disorders[END_REF][START_REF] Jan | Rapid cycling in severely multidisabled children: A form of bipolar affective disorder?[END_REF][START_REF] Castro-Silva | Melatonin improves sleep and reduces nitrite in the exhaled breath condensate in cystic fibrosis--a randomized, double-blind placebo-controlled study[END_REF], including disorders associated with delayed phase sleep syndrome [START_REF] Dahlitz | Delayed sleep phase syndrome response to melatonin[END_REF][START_REF] Mundey | Phase-dependent treatment of delayed sleep phase syndrome with melatonin[END_REF][START_REF] Oldani | Melatonin and delayed sleep phase syndrome: Ambulatory polygraphic evaluation[END_REF], periodic sleep disorders in blind patients [START_REF] Lapierre | Melatonin treatment of a non-24-hour sleep-wake cycle in a blind retarded child[END_REF][START_REF] Folkard | Melatonin stabilises sleep onset time in a blind man without entrainment of cortisol or temperature rhythms[END_REF][START_REF] Arendt | Synchronisation of a disturbed sleep-wake cycle in a blind man by melatonin treatment (letter)[END_REF][START_REF] Sack | Melatonin administration to blind people: Phase advances and entrainment[END_REF] and sleep and behavioral disorders in children with severe brain damage [START_REF] Palm | Correction of non-24-hour sleep/wake cycle by melatonin in a blind retarded boy[END_REF][START_REF] Jan | The treatment of sleep disorders with melatonin[END_REF][START_REF] Jan | Use of melatonin in the treatment of paediatric sleep disorders[END_REF][START_REF] Jan | Rapid cycling in severely multidisabled children: A form of bipolar affective disorder?[END_REF]. In a randomized double-blind, placebo-controlled study [START_REF] Castro-Silva | Melatonin improves sleep and reduces nitrite in the exhaled breath condensate in cystic fibrosis--a randomized, double-blind placebo-controlled study[END_REF], melatonin was shown to improve sleep efficiency and sleep latency [START_REF] Castro-Silva | Melatonin improves sleep and reduces nitrite in the exhaled breath condensate in cystic fibrosis--a randomized, double-blind placebo-controlled study[END_REF] in cystic fibrosis (CF) patients, a chronic progressive disorder characterized by repeated episodes of respiratory distress and sleep disturbances [START_REF] Castro-Silva | Melatonin improves sleep and reduces nitrite in the exhaled breath condensate in cystic fibrosis--a randomized, double-blind placebo-controlled study[END_REF].

However, most of the clinical trials involving melatonin exploit its anti-oxidant properties.

Melatonin was shown to reduce nitrite levels in exhaled breath condensate in CF patients [START_REF] Castro-Silva | Melatonin improves sleep and reduces nitrite in the exhaled breath condensate in cystic fibrosis--a randomized, double-blind placebo-controlled study[END_REF].

Sepsis is a severe condition associated with a significant imbalance of the intracellular redox state resulting from an increased production of oxidant species and a decrease in endogenous antioxidant defenses. In critically ill patients, sources of oxidative stress include the mitochondrial respiratory electron transport chain [START_REF] Fredriksson | Derangements in mitochondrial metabolism in intercostal and leg muscle of critically ill patients with sepsis-induced multiple organ failure[END_REF], the respiratory burst associated with neutrophil activation [START_REF] Bogdan | Reactive oxygen and reactive nitrogen intermediates in innate and specific immunity[END_REF], and arachidonic acid metabolism [START_REF] Rinaldi | Antioxidant therapy in critically septic patients[END_REF]. Melatonin exerts well-documented protective effects against septic shock in both animals and humans. Melatonin was investigated in a prospective clinical trial regarding antioxidant therapy to inhibit the action of nitro-oxidative stress [133]. It was demonstrated that nitric oxide (NO) plays an important role in septic shock, an acute inflammatory response [START_REF] Bubenik | Prospects of the clinical utilization of melatonin[END_REF]. Indeed, melatonin improves the survival of mice with LPS-induced shock by reducing NO synthesis via its antioxidant and anti-apoptotic properties [START_REF] Carrillo-Vico | Beneficial pleiotropic actions of melatonin in an experimental model of septic shock in mice: regulation of pro-/anti-inflammatory cytokine network, protection against oxidative damage and anti-apoptotic effects[END_REF]. It was demonstrated that antioxidant treatment improves respiratory syncytial virus (RSV)-induced pulmonary inflammation. Interestingly, the administration of melatonin reverses the oxidative stress induced by RSV by reducing NO and hydroxyl radical (OH) levels and by increasing glutathione (GSH) level and SOD activity [START_REF] Huang | Inhibitory effect of melatonin on lung oxidative stress induced by respiratory syncytial virus infection in mice[END_REF]. At the same time, melatonin inhibits the production of pro-inflammatory cytokines such as TNF-α improving RSV-induced lung inflammatory injury in mice.

Melatonin prevents multiple organ failure, circulatory failure, and mitochondrial damage in model of experimental sepsis [137], and reduces mortality in septic children together with lipid peroxidation as an index of inflammation [START_REF] Gitto | Effects of melatonin treatment in septic newborns[END_REF]. Melatonin was shown to improve clinical outcome in terms of infection and organ failure in randomized clinical trials, thus being a promising antioxidant molecule in antioxidant therapy to combat sepsis [START_REF] Rinaldi | Antioxidant therapy in critically septic patients[END_REF][133][START_REF] Bubenik | Prospects of the clinical utilization of melatonin[END_REF]137].

Administration of melatonin to humans and animals at both physiological and pharmacological concentrations is essentially non-toxic; it can be easily synthesized in a pharmacologically pure form or extracted by natural sources; it possesses outstanding versatility in reducing oxidative stress and inflammation. Altogether, these features make of melatonin a potential therapeutic tool to improve human health.

Conclusions and perspectives

Commonly used anti-inflammatory agents hardy distinguish between physiological and pathological inflammation; the indiscriminate use of anti-inflammatory agents can lead to a deregulation of the inflammatory response, with the risk to fail to fight pathogens or injuries. The differential pro-and anti-inflammatory roles of melatonin might promote, regulate, and counteract inflammation simultaneously. Indeed, melatonin, as an anti-inflammatory molecule, could reduce oxidative tissue injuries by its anti-oxidant properties and selectively inhibit the late/chronic phases of the inflammatory response. Furthermore, melatonin could act on early phases of inflammation by stimulating proinflammatory mediators such as arachidonic acid and 5-HETE via activation of PLA2 and 5-LOX, respectively [START_REF] Radogna | Lipoxygenase-mediated pro-radical effect of melatonin via stimulation of arachidonic acid metabolism[END_REF]. Interestingly, effects of 5-LOX extinguish within 2-3 h, since a down-regulation of the same enzymes is also triggered by melatonin as a later event induced by different mechanisms [START_REF] Li | Negative regulation of cytosolic phospholipase A(2) by melatonin in the rat pineal gland[END_REF][START_REF] Steinhilber | The nuclear receptor for melatonin repress 5lipoxygenase gene expression in human B lymphocytes[END_REF]. Thus, melatonin might facilitate the promotion of the inflammatory response, at the same time limiting it by providing to its resolution via down-regulation of 5-LOX and/or PLA2.

Altogether melatonin could then avoid complications of chronic inflammation. Freshly explanted blood monocytes require LOX-derived signals and also ROS to maximize in vitro activation and viability, two events that require the over-expression of Bcl-2 [START_REF] Cristofanon | Oxidationdependent maturation and survival of explanted blood monocytes via Bcl-2 up-regulation[END_REF]. The role of LOX in maintaining cell viability is not unusual; 5-LOX expression is required to maintain viability of some tumor cells; in addition, in EBV-converted B lymphocytes, 5-LOX inhibition is a rapid and efficient mean of killing EBV+ cells [START_REF] Belfiore | Involvement of 5-lipoxygenase in survival of Epstein-Barr virus (EBV)-converted B lymphoma cells[END_REF].

It has been long debated that melatonin in some instances may exert a pro-apoptotic role [START_REF] Sainz | Melatonin and cell death: differential actions on apoptosis in normal and cancer cells[END_REF]; the mechanisms and rationale of this effect are unclear. However, upon Bcl-2 silencing, melatonin acquires pro-apoptotic abilities [START_REF] Radogna | Melatonin antagonizes the intrinsic pathway of apoptosis via mitochondrial targeting of Bcl-2[END_REF], indicating that the very finely regulated changes in Bcl-2 levels during lymphocyte maturation may confer different susceptibility to melatonin, modulating a pro-or anti-apoptotic effect according to the intracellular amount of Bcl-2.

Thus, melatonin could promote acute inflammation recruiting leukocytes via an indirect prooxidant effect consisting of 5-LOX activation and promotion of inflammatory products, and prolonging the lifespan of the recruited leukocytes; it may also prevent chronic inflammation by its anti-oxidant ability. This includes a reduction of oxidative damage in the injured tissue as well as the inhibition of pro-inflammatory mediators; break the vicious cycle of oxidation/leukocyte recruitment, and promoting leukocyte apoptosis (Figure 4).

  PLEIOTROPIC MOLECULE REGULATING INFLAMMATION Flavia Radogna, Marc Diederich° and Lina Ghibelli* Dipartimento di Biologia, Universita" di Roma Tor Vergata ° Laboratoire de Biologie Moléculaire et Cellulaire du Cancer, Hôpital Kirchberg, 9 rue Edward Steichen, L-2540 Luxembourg, LuxembourgAddress correspondence to: Prof Lina Ghibelli, Dipartimento di Biologia, Universita` di Roma "Tor Vergata", Via della Ricerca Scientifica, I-00133 Rome, Italy. E-mail: ghibelli@uniroma2.it

  depending on the intra-and extra-cellular environment. Even though the mechanisms through

6 -

 6 A c c e p t e d M a n u s c r i p t -Leucocytes possess the enzymatic machinery necessary to synthesize melatonin.

  bacterial effect in the injured area in spite of cellular injuries. To this purpose, the organism generated cellular resistance strategies contributing to extend leukocyte lifetime by avoiding or delaying cell death mechanisms.

  Altogether these investigations open new important avenues for the pharmacological use of melatonin in inflammation as the stimulation of different targets, combined with differential cellular sensitivities generates a variety of effects witnessing sophisticated regulatory mechanisms.

Figure 1 :Figure 2 :

 12 Figure 1: Biosynthesis of melatonin

Figure 3 :

 3 Figure 3: Dual effects of melatonin on lipoxygenase. Melatonin both down-regulates lipoxygenase gene expression through RZR-beta (A) and activates 5-LOX via PLA2 activation via calmodulindependent signaling (B).

Figure 4 :

 4 Figure 4: Pleiotropic effects of melatonin on different steps of inflammation. Summary of the multiple effects that melatonin exerts at different steps of the inflammatory response, indicating a pro-inflammatory role at an early phase, and an antagonist role at later phases. This evokes a smart behavior where melatonin may favor the inflammatory healing processes while contrasting pathologically chronic or deregulated inflammation, thus potentially being an ideal compound to treat inflammatory disturbances.

Acknowledgements

F.R. is a recipient of a postdoctoral "Recherches Scientifiques Luxembourg (RSL)" grant. M.D."s lab is financed by "Recherches Scientifiques Luxembourg (RSL)", ""Recherche Cancer et Sang"" foundation, by ""Een Haerz fir kriibskrank Kanner"" association, by the Action Lions ""Vaincre le Cancer"" association and by Télévie Luxembourg.

C. RZR ROR

*Graphical Abstract


" Christophe Gane

Hézode

MD Christophe Hézode
email: christophe.hezode@hmn.aphp.fr

Oral combination therapy with a nucleoside polymerase inhibitor (RG7128) and danoprevir for chronic hepatitis C genotype 1 infection (INFORM-1): a randomised, double-blind, placebo-controlled, dose-escalation trial

In the last decade, insights into the virology of HCV have unraveled several targets for potential novel therapeutics that, unlike interferon (IFN)- and ribavirin, are specifically targeted to HCV. Several new drug therapies such as protease and polymerase inhibitors, designated as direct-acting antivirals (DAAs) have reached clinical development [START_REF] Poordad | Big changes are coming in hepatitis C[END_REF][START_REF] Asselah | New direct-acting antivirals combination fot the treatment of chronic hepatitis C[END_REF]. Gane and colleagues have recently published in the Lancet the results of the first study with a combination of two (DAAs) without interferon (IFN) or ribavirin in patients with chronic hepatitis C. The aim of the proof-of-concept INFORM-1 trial was to assess the efficacy and safety of the combination of two drugs in development, danoprevir, an NS3/4A protease inhibitor, and RG7128, a nucleoside analogue inhibitor of HCV RNA-dependent RNA polymerase, over two weeks of administration. In this randomised, double-blind, placebocontrolled trial, 88 patients infected with HCV genotype 1 were enrolled into one of seven treatment arms, randomised to different doses and schedules of study drugs or placebo for up to 13 days. Then, patients were rolled over into the current standard of care (SOC), i.e. pegylated interferon (PEG-IFN) and ribavirin (RBV). The vast majority of patients had never been treated before, whereas 16 of them had failed to eradicate HCV with SOC. The main finding of the study was the demonstration of a substantial, sustained, dose-dependent HCV RNA level reduction, whatever the treatment group. The viral level reduction reached -5 log on average after 14 days of combination with the highest drug doses i.e. 1000 mg b.i.d of RG7128 and 900 mg b.i.d. of danoprevir. Undetectable HCV-RNA (<15 IU/mL) was achieved in 44% of patients overall. The antiviral efficacy was similar in treatment-naïve patients and in patients who failed to eradicate HCV after a first course with PEG-IFN and RBV, including null-responders included in the highest dose group. No evidence of emergence of HCV variants resistant to either compound was reported during the short-term administration of the drugs. Treatment was well tolerated and no serious adverse event, dose modification or premature discontinuation were reported over the two weeks of administration.

Virtually any step of the HCV lifecycle can be a target for HCV inhibitors. The most successful approach so far has been targeting the HCV protease. The triple combination of PEG-IFN, RBV and a first-generation protease inhibitor (telaprevir or boceprevir) will increase the sustained virologic response (SVR) rates from 40-44% to 68-75% in treatmentnaïve patients infected with HCV genotype 1 [START_REF] Jacobson | Telaprevir in combination with peginterferon and ribavirin in genotype 1 HCV treatment-naïve patients: final results of phase 3 advance study[END_REF][START_REF] Poordad | Boceprevir (BOC) combined with peginterferon alfa-2b/ribavirin (P/R) for treatmentnaïve patients with hepatitis C virus (HCV) genotype (G) 1: sprint-2 final results[END_REF]. This treatment is expected to become the new SOC at the end of 2011-beginning of 2012 in treatment-naïve and treatment-experienced patients infected with genotype 1. However, this therapy will be not be optimal for patients who are intolerant or have contra-indications to PEG-IFN or RBV, including for instance patients with decompensated cirrhosis or hemoglobinopathies. In the phase 3 registration trials, more than 10% of the carefully selected patients discontinued triple combination therapy prematurely due to adverse events. Moreover, the efficacy of the triple combination will be limited in treatment-experienced patients who did not respond to PEG-IFN and ribavirin by a more than 1 log drop in HCV RNA levels at week 4, with a high risk of treatment failure and the selection of HCV variants resistant to protease inhibitors [START_REF] Mchutchison | Telaprevir for previously treated chronic HCV infection[END_REF][START_REF] Bacon | HCV respond-2 final results: high sustained virologic response among genotype 1 previous non-responders and relapsers to peginterferon/ribavirin when re-treated with boceprevir plus pegintron (peginterferon alfa-2b)/ribavirin [abstract[END_REF].

Finally, triple therapy will most likely not be able to eradicate HCV in a number of so-called "difficult-to-treat" patients. Thus, PEG-IFN and/or RBV intolerant or poorly responsive patients could be the main beneficiaries of interferon-sparing HCV treatment regimens, and one cannot exclude that IFN-free regimens will become SOC first-line treatment for all patients with chronic HCV infection. Although the present study met the short-term safety objective and direct interactions between the two drugs could be considered unlikely, it presents some limitations. Due to the short duration of administration of the study drugs, the long-term safety profile and the risk of late rebound due to viral resistance to the combination remain unknown. The goal of antiviral treatment is to cure HCV infection, as witnessed by the sustained virological response, characterized by an undetectable HCV RNA 6 months after the end of therapy. Because the patients were all treated with the combination of PEG-IFN and ribavirin after the two-week IFN-free regimen, the fact that the observed early pronounced antiviral activity of the combination of two DAAs could lead to HCV eradication has not been proven. However, interestingly, the early kinetics of HCV RNA levels with the DAA combination appeared to be similar to those reported in a previous study with a similar population receiving danoprevir in combination with PEG-IFN and ribavirin. The initial HCV RNA decline was rapid, especially during the first two days (first phase decline: -3.5 log on average with the highest drug doses in treatment-naïve patients), corresponding to the clearance of free HCV virions.

The second slope was slower (HCV-RNA decline: -1.0 log per week on average), related to the loss of infected hepatocytes (Figure 1). Based on an estimated total body viral burden of 10 11 virions, 8 to 12 weeks of DAA treatment could be sufficient to eradicate HCV infection in most patients, provided that no relapse occurs as a result of resistant variant selection. The possibility to cure HCV infection with DAA combinations without IFN (with or without ribavirin) should be assessed in future studies with longer treatment durations in both treatment-naïve and -experienced patients, as all-oral, IFN-free regimens should be effective in both patient populations.

Current and future trials will likely determine whether the IL28B genotype and IP-10 levels influence SVR when patients are treated with DAA combinations without IFN. The impact of RBV on the efficacy and safety of DAA combinations has to be established. Based on the observation of higher relapse and viral breakthrough rates in the RBV-sparing treatment arms in the telaprevir plus SOC studies [START_REF] Mchutchison | Telaprevir for previously treated chronic HCV infection[END_REF][START_REF] Hézode | Telaprevir and peginterferon with or without ribavirin for chronic HCV infection[END_REF] and on the fact that RBV could accelerate the second slope of viral decline and prevent breakthrough due to resistance when administered in combination with two DAAs with a low genetic barrier to resistance [START_REF] Zeuzem | Dual, triple, and quadruple combination treatment with a protease inhibitor (GS-9256) and a polymerase inhibitor (GS-9190) alone and in combination with ribavirin (RBV) or PEGIFN/RBV for up to 28 days in treatment naïve , genotype 1 HCV subjects[END_REF], the impact of RBV on efficacy and safety of combination DAA therapy has to be established.

Although telaprevir has shown potent antiviral activity against HCV genotype 2, it has no effect on HCV genotype 3, suggesting that PEG-IFN and RBV will remain the SOC for non HCV genotype 1 patients [START_REF] Foster | Activity of telaprevir alone or in combination with peginterferon alfa-2a and ribavirin in treatment-naïve, genotype 2 and 3, hepatitis-C patients: final results of study C209[END_REF]. However, other protease inhibitors, as well as nucleoside/nucleotide analogues and cyclophillin inhibitors, have shown antiviral potency against genotypes other than 1. Thus, IFN-sparing combination regimens could be studied in other patient populations than those included in INFORM-1.

Several trials of combination DAAs are ongoing in treatment-naïve or experienced patients. All studies include protease inhibitor, combined with either a NS5A, non-nucleoside NS5B, nucleoside NS5B inhibitor. The second DAA must be chosen carefully, as recent data suggest that the genetic barrier of combination therapy with a first-generation protease inhibitor and a non-nucleoside NS5B or an NS5A inhibitor is not higher than that of protease inhibitor monotherapy [START_REF] Zeuzem | Dual, triple, and quadruple combination treatment with a protease inhibitor (GS-9256) and a polymerase inhibitor (GS-9190) alone and in combination with ribavirin (RBV) or PEGIFN/RBV for up to 28 days in treatment naïve , genotype 1 HCV subjects[END_REF][START_REF] Lok | Combination therapy with BMS-790052 and BMS-650032 alone or with PegIFN/RBV results in undetectable HCV RNA through 12 weeks of therapy in HCV genotype 1 null responders[END_REF]. In contrast, the absence of occurrence of breakthroughs during this trial is probably related to the high barrier resistance of the danoprevir and RG7128 combination.

In conclusion, this proof-of-concept INFORM-1 trial has shown that the combination of danoprevir and RG7128 for up to 13 days results in a substantial biphasic HCV-RNA decline, without selecting HCV variants and with a satisfactory safety profile in both treatment-naïve or treatment-experienced patients infected with HCV genotype 1. Further trials with these drugs or other DAAs, with or without ribavirin are now needed to assess whether IFN-free regimens may provide high cure rates in the future.

  The main objectives of a regimen combining DAAs without IFN or ribavirin should be: a) to improve antiviral efficacy without increasing toxicity; b) to prevent the development of resistance by combining DAAs targeting different elements of the HCV life cycle and without cross-resistance; and c) to avoid drug-drug interactions without impairing safety.

Figure 1 :

 1 Figure 1: Early viral kinetic profile of danoprevir and RG7128 combination.