Beneficial actions of melatonin in the management of viral infections: a new use for this "molecular handyman"?

Melatonin (N-acetyl-5-methoxytryptamine) is a multifunctional signaling molecule that has a variety of important functions. Numerous clinical trials have examined the therapeutic usefulness of melatonin in different fields of medicine. Clinical trials have shown that melatonin is efficient in preventing cell damage under acute (sepsis, asphyxia in newborns) and chronic states (metabolic and neurodegenerative diseases, cancer, inflammation, aging). The beneficial effects of melatonin can be explained by its properties as a potent antioxidant and antioxidant enzyme inducer, a regulator of apoptosis and a stimulator of immune functions. These effects support the use of melatonin in viral infections, which are often associated with inflammatory injury and increases in oxidative stress. In fact, melatonin has been used recently to treat several viral infections, which are summarized in this review. The role of melatonin in infections is also discussed herein.

AIM2‐like receptors

Aleutian mink disease virus

activating protein‐1

activation transcription factor 2

blood–brain barrier

catalase

death‐inducing signaling complex

encephalomyocarditis virus

granulocyte‐macrophage colony‐stimulating factor

glutathione peroxidase

glutathione‐s‐transferase

human papillomavirus

interferon‐induced protein with tetratricopeptide

inducible NO synthase

interferon regulatory factor 3

interferon regulatory factor 7

interferon‐stimulated genes

Janus kinase

monocyte chemotactic protein‐1

malondialdehyde

murine leukemia virus

mitochondrial permeability transition pore

nuclear factor kappa B

natural killer cells

natural killer T cells

Nod‐like receptors

nuclear factor erythroid 2

oligoadenylate synthetases

pathogen‐associated molecular patterns

programmed cell death

plasmacytoid dendritic cells

dsRNA‐activated protein kinase

pattern recognition receptors

regulated upon activation, normal T cell expressed and secreted

rabbit hemorrhagic disease virus

RIG‐I‐like receptors

Sendai virus

Semliki Forest virus

superoxide dismutases

tickborn encephalitis virus

transforming growth factor‐β

type 1 T helper cell

type 2 T helper cell

Toll‐like receptors

tumor necrosis factor receptor

Venezuelan equine encephalomyelitis

Venezuelan equine encephalomyelitis virus

vesicular stomatitis virus

West Nile virus

xanthine oxidase

INTRODUCTION

The methoxyindole melatonin (N‐acetyl‐5‐methoxytryptamine) is a secretory product of the pineal gland. It was first reported as a skin lightening agent in amphibians 1, 2. Further investigations showed that another function, supported by its direct effects in regions containing high densities of melatonin receptors, such as the circadian pacemaker (the suprachiasmatic nucleus) and the pars tuberalis, is to regulate and reset circadian rhythms as well as to be involved in the measurement of day length, an environmental variable used for seasonal timing of reproduction, metabolism and behavior in species responding to photoperiodic changes 3, 4, 5, 6, 7.

In recent decades, melatonin has been reported to possess numerous additional functions and act in neural and non‐neural tissues or cells that express melatonin receptors that are at lower densities than in the suprachiasmatic nucleus. Thus, melatonin is involved in sleep initiation, vasomotor control, anti‐excitatory actions, immunomodulation including possessing anti‐inflammatory properties, antioxidant actions, and actions on energy metabolism, influences on mitochondrial electron flux, regulation of the mitochondrial permeability transition pore (mtPTP), and mitochondrial protection against free radicals 8, 9, 10, 11, 12, 13. Deficiencies in melatonin production or melatonin receptor expression and decreases in melatonin levels (such as those that occur during aging) are likely to contribute to numerous dysfunctions 14, 15, 16. In fact, several clinical trials have shown that melatonin is efficient in preventing cell damage under acute (sepsis, asphyxia in newborns) and chronic states (metabolic and neurodegenerative diseases, cancer, inflammation, aging) 17, 18, 19, 20, 21, 22. In humans, the efficacy of melatonin as a treatment of ocular diseases, cardiovascular diseases, sleep disturbances and several other pathologies, as well as a complementary treatment in anesthesia, haemodialysis, in vitro fertilization and neonatal care, has been assessed and reported to be beneficial 23. Likewise, melatonin reduces the toxicity and increases the efficacy of a large number of drugs whose side effects are well documented 24.

The beneficial effects of melatonin are explained by its properties as a potent antioxidant, a modulator of apoptosis and a positive regulator of immune functions 25, 26, 27, 28, 29. These actions suggest the potential to treat viral infections, which usually cause inflammatory injury and elevated oxidative stress 30, 31. A number of reports examining the ability of melatonin to protect against viral infections have been published, as summarized in the following section.

FIRST EVIDENCE RELATED TO THE ABILITY OF MELATONIN TO PROTECT AGAINST TO VIRAL INFECTIONS

Encephalomyocarditis virus (EMCV) is a highly pathogenic and aggressive virus that causes encephalitis and myocarditis in rodents. Administration of melatonin prevented paralysis and death of mice infected with sublethal doses of EMCV 32. Melatonin also has a protective effect in mice infected with Semliki Forest virus (SFV), a classic encephalitis arbovirus, that invades the CNS and whose replication in the mouse brain eventually leads to death. Melatonin administration not only reduced the death rate but also significantly postponed the onset of the disease. Furthermore, the level of virus in the blood in melatonin‐treated mice was lower than in non‐treated mice 33. Although attenuated West Nile virus (WNV) strain WN‐25 is an encephalitis virus that does not invade the brain and does not normally cause encephalitis, exposure of mice to various stressful stimuli induces WN‐25 encephalitis. Melatonin counteracts the immunodepressive effect of stress exposure and prevents the stress‐related encephalitis and death of WN‐25 infected mice 33.

Venezuelan equine encephalomyelitis (VEE) is an important human and equine disease caused by VEE virus (VEEV), a mosquito‐borne organism. Outbreaks have occurred in northern South America from the 1920s to the 1970s with thousands of people and horses, donkeys and related species being infected. Mice have been used as an animal model for this condition, because VEEV‐infected mice show excitation and hypermotility followed by hypomotility, paralysis, coma and death. Melatonin administration protects mice infected with VEEV by decreasing the virus load in brain and serum, reducing mortality rates, delaying the onset of the disease and deferring the time to death. Furthermore, in surviving mice treated with melatonin, the VEEV‐mediated IgM antibody titres are highly elevated 34.

Aleutian mink disease is a natural condition caused by persistent infection with the Aleutian mink disease virus (AMDV). Animals in the progressive state of the disease show a marked hypergammaglobulinemia, because of high titers of non‐neutralizing ADMV antibodies. This is thought to cause lesions in the kidney, liver, lungs and arteries. Melatonin implants reduced mortality in ADMV‐infected mink 35.

The findings in these reports document the ability of the melatonin to protect against viral infections [Table 1]. The potential protective mechanisms include melatonin acting as a free radical scavenger, an antioxidant enzyme inducer, a positive regulator of immune functions and an inhibitor of inflammation, as well as a regulator of programmed cell death (PCD) [Table 2].

Table 1

First evidence related to the ability of melatonin to protect against viral infections

Virus	Animals	Doses of melatonin	Effects	Ref.
EMCV	25 female 2‐3‐months‐old BALB/cj mice	1 µg i.p./mouse daily for 10 days	Prevention of paralysis and death of infected mice	32
SFV	18 Charles River outbred ICR female mice (CD1)	500 µg/kg s.c. daily, 3 days before until 10 days after virus inoculation	Reduction of the death rate	33
			Delay of the onset of the disease
	10 Charles River outbred ICR female mice (CD1)	500 µg/kg s.c. daily, 3 days before until 10 days after virus inoculation	Decrease the virus load in blood	33
WN‐25*	16 Charles River outbred ICR female mice (CD1)	5 µg/mouse s.c. daily, 2 days before until 8 days after virus inoculation	Counteracts the immunodepressive effect of stress exposure	33
			Prevention of the stress‐related encephalitis
			Prevention of the death of infected mice
VEEV	25 male albino mice (NMRI‐ IVIC strain)/group	250–500 µg–1 mg/kg s.c. daily, 3 days before until 10 days after inoculation	Reduction of mortality rates	34
			Delay of the onset of the disease
			Deferring of the time to death
	6 male albino mice (NMRI‐ IVIC strain)	500 µg/kg s.c. daily, 3 days before until 10 days after inoculation	Decrease the virus load in brain Decrease the virus load in serum	34
	3 male albino mice (NMRI‐ IVIC strain)/group	250–500 µg/kg s.c. daily, 3 days before until 10 days after inoculation	Increase the VEEV‐mediated IgM antibody titres	34
AMDV	90 wild type (demi‐buff or demi strain) minks 6000 male and female demi strain minks 3000 male and female demi and mahogany strains of kit minks	Silastic implants (0.65 cm length, 0.21 cm diameter) containing 2.7 mg melatonin crystals homogeneously suspended in medical grade silastic polymer	Reduction of mortality rates	35

EMCV, encephalomyocarditis virus; SFV, Semliki Forest virus; VEEV, Venezuelan equine encephalomyelitis virus; AMDV, Aleutian mink disease virus; i.p., intraperitoneal; s.c., subcutaneal.

an atenuated West Nile virus strain

Table 2

Effects of melatonin in protecting against viral infections

Properties	Virus	Animal/cell cultures	Effects of melatonin administration	Ref.
Free radical scavenger	VEEV	Murine splenocytes	Reduction of NO• concentrations in tissue	105
		Murine neuroblastoma	Decrease of both NO• and lipid peroxidation	106
		Mice	Reduces nitrite concentrations in the brain and serum	107
			Lowering lipid peroxidation products
	RSV	Mice	Reduction of acute lung oxidative injury	31
			Suppression of MDA, NO• and •OH generation
			Restoration of GSH and SOD levels in the lungs
Antioxidant enzyme inducer	RHDV	Rabbits	Restoration of activity and mRNA expression of GPx, GST and Mn‐SOD	109
			Rise in protein expression of Nrf2
Regulator of immune functions	MLV	Mice	Prevention of reduction in B‐ and T‐cell proliferation	156
			Prevention in Th1 cytokine secretion
			Prevention of overproduction of Th2 cytokines and TNF‐α
	VEEV	Mice	Stimulation of endogenous production of IL‐1β in brain	158
			Reduction of the concentration of TNF‐α in brain
			Stimulation of endogenous production of IFN‐γ, IL‐1β, and TNF‐α in serum	159
	RSV	Mouse macrophages	Decrease of TLR3‐mediated downstream gene expression	170
Regulator of PCD	RHDV	Rabbits	Reduction of Bax expression	179
			Reduction of cytosolic cytochrome c release
			Increased expression of Bcl‐2 and Bcl‐xL
			Inhibition of caspase‐9 activity
			Reduction in caspase‐8 activity
			Reduction in TNF‐R1 and JNK expression
			Increased expression of c‐FLIP

VEEV, Venezuelan equine encephalomyelitis virus; RHDV, rabbit hemorrhagic disease virus; MLV, murine leukemia virus.

MELATONIN AS A FREE RADICAL SCAVENGER AND ANTIOXIDANT ENZYME INDUCER IN VIRAL INFECTIONS

Free radicals are molecules formed naturally during many metabolic processes. They contain an unpaired electron in their valence orbital that makes them unstable and reactive. These reactive agents damage essential molecules in cells including lipids, proteins and DNA 36, 37. Among these reactants, the superoxide anion radical (O2•−), nitric oxide (NO•) and especially their derivatives, the hydroxyl radical (•OH) and peroxynitrite (ONOO−), are highly biologically damaging elements produced in the host during microbial infections 38, 39, 40, 41. Phagocytes, such as neutrophils and macrophages are assumed to be the major generators of free radicals. Elevated levels of O2•− are generated by both phagocyte NADPH oxidase and xanthine oxidase (XO) during viral infections 42, 43, 44, 45, 46. O2•− reduces ferric iron to ferrous iron, which catalyzes the Fenton reaction and generates •OH from hydrogen peroxide. ONOO− is formed by the coupling of O2•− and NO•.

Overproduction of NO• is primarily caused by activation of inducible NO synthase (iNOS), which is usually expressed by inflammatory phagocytes and other cell types (e.g. epithelial and neuronal cells) 37, 38, 40, 47. iNOS is regulated by cytokine‐dependent mechanisms in HIV‐1, HBV and HCV infections 48, 49, 50, 51, as well as in a variety of experimental viral infections in rats and mice, including neurotropicviruses (Borna disease virus, HSV‐1 and rabies virus), and pneumotropic and cardiotropic viruses (influenza virus, SeV and coxsackie virus) 52, 53, 54, 55, 56, 57, 58, 59.

Although IFN‐γ is the major cytokine inducing iNOS and NO• overproduction in the pathogenesis of these viral infections, iNOS expression is downregulated by IL‐4, IL‐10 and transforming growth factor‐β (TGF‐β) 60, 61, 62. IFN‐γ is known to be associated with type 1 helper T cell (Th1) responses, and IL‐4 and IL‐10 are induced by type 2 helper T cell (Th2) responses; NO• biosynthesis catalyzed by iNOS is precisely regulated by a polarized Th1–Th2 balance. In other viral diseases, viral replication or viral components directly induce iNOS without mediation by pro‐inflammatory cytokines. Thus, the HIV envelope glycoprotein gp41 triggers iNOS expression in human astrocytes and murine cortical brain cells in culture 63, 64. RSV directly upregulates iNOS in human type 2 alveolar epithelial cells (A549 cells) 65.

Free radicals are produced to eliminate the pathogenic agent or to kill the virus‐infected cells by a non‐specific response. Thus, antiviral effects of NO• have been described for some DNA viruses such as murine poxvirus (ectromelia virus) and herpes viruses including HSV, EBV and some RNA viruses such as Coxsackie virus 66, 67, 68, 69, 70, 71. The toxic oxygen and nitrogen‐based reactants, unfortunately, cannot discriminate between exogenous invading pathogens and the host cells themselves, and therefore, they also damage the host. To minimize such self‐damage during the elimination of pathogens, the host employs several primitive tactics; it uses recruited phagocytes for the physical containment of pathogens in infectious foci. Most bacteria, for example, can be phagocytosed and confined to septic foci, which are typically abscesses or granulomas. Under these conditions, free radicals can affect bacteria rather selectively with the surrounding normal tissue remaining mostly intact.

In viral infections, in contrast, free radical mediators cause non‐specific oxidative/nitrosative damage in virus‐infected tissue and produce oxidative stress; this occurs when the virus cannot be confined to limited areas by the non‐specific host defense 56, 58, 72. Thus, NO• has appreciable antiviral actions on several types of viruses including ortho‐ and paramyxovirus, murine vaccinia virus, coronavirus (mouse hepatitis virus), lymphocytic choriomeningitis virus, murine EMCV, tickborn encephalitis virus (TBE‐V) 73, 74, 75, 76, 77, 78; also, NO• and its derivatives, especially ONOO‐, can be considered pathogenic in some viral infections. Indeed, NO• inhibition or lack of NO• generation reduces the pathological consequences of viral pneumonia in mice caused by influenza virus, SeV and HSV‐1, HSV‐1‐induced encephalitis in rats, EMCV‐induced carditis and diabetes, and murine encephalitis induced by flavivirus (Murray Valley encephalitis virus, TBE‐V) 55, 57, 74, 78, 79, 80, 81, 82. A similar pathogenicity with a lack of antiviral effects has been observed for O2•‐ in several experimental models of virus‐induced pneumonia including those caused by influenza virus and CMV 43, 44, 45, 56, 72, 83, 84.

HCV‐induced oxidative stress is emerging as a key step and a major initiator in the development and the progression of liver damage 85. NS3, one of the non‐structural proteins of HCV, was reported to induce reactive oxygen species by NADPH oxidase in neutrophils 86. High‐risk human papilloma virus (HPV), which causes cervical cancer, promotes iNOS‐dependent DNA damage, leading to dysplastic changes and carcinogenesis 87.

Epstein–Barr virus is a herpes virus that infects the majority of the world population, generally during childhood; it has been linked to the genesis of a number of lymphoproliferative diseases and neoplasia such as the African Burkitt lymphoma, nasopharyngeal carcinoma or gastric carcinoma. Early stages of EBV infection generate oxidative stress either in B lymphocytes or in epithelial cells, so contributing to pathology 88. Influenza A virus causes a respiratory disease, which ranges from mild upper respiratory tract illness with or without fever to severe complications such as pneumonia. The latter disease results in respiratory failure, acute respiratory distress syndrome, multi‐organ failure and even death. An abrupt increase in O2•− production occurs during phagocytosis, which induces injury in non‐infected cells. These O2•−‐mediated pathways contribute to a portion of the extensive tissue injury observed during severe influenza‐associated complications 56.

To protect themselves against free radical‐mediated damage, cells have developed an antioxidant defense that includes enzymatic and non‐enzymatic mechanisms. Free radical generation and a functionally efficient antioxidant defense system must be in equilibrium to avoid cellular damage caused by radicals and their derivatives. Enzymes involved in the elimination of free radicals include the superoxide dismutases (SOD), catalase (CAT) and glutathione peroxidase (GPx). In addition to the enzymatic antioxidant system, organisms possess non‐enzymatic free radical scavengers, which directly remove toxic reactants because of their electron donating ability. The best known non‐enzymatic antioxidants are vitamin E (α‐tocopherol), vitamin C (ascorbate), glutathione (GSH), β‐carotene and, as recently described, melatonin 25. Several radical scavengers have been efficacious in ameliorating the severity of viral diseases. N‐acetylcysteine, a GSH precursor, inhibits HIV in vitro 89 as did the natural thiol antioxidant, alpha‐lipoic acid 90. Glutathione administration to HIV seropositive individuals by aerosol treatment can correct the glutathione deficiency 91. The combination of several antioxidants with antiviral drugs synergistically reduces the lethal effects of influenza virus infections 92. Thus, any agent that functions as a direct radical scavenger and also stimulates antioxidative enzymes could have utility in the treatment of patients with severe complications of viral infections.

Melatonin is a powerful and effective •OH scavenger, which provides protection against oxidative damage of cell components. It also scavenges the peroxyl radical to a lesser degree generated during lipid peroxidation with an activity that, in some situations, is reportedly greater than that of vitamin E 22, 93, 94, 95, 96. Also, melatonin directly detoxifies the ONOO− and possibly peroxynitrous acid (ONOOH) 97. In vivo, melatonin stimulates several antioxidative enzymes including GPx, CAT and SOD, thereby potentiating its antioxidant properties 98, 99, 100, 101. Melatonin can cross anatomical barriers, including the placenta and the blood–brain barrier 102, 103, and easily enter cells 104.

Splenocytes infected with VEEV generated less of NO•, when treated with melatonin; this finding suggests that the indoleamine protected mice infected with the VEEV by a mechanism involving a reduction in NO• concentrations in tissue 105. Elevated production of NO• and lipid peroxidation products were also found in supernatants and cellular elements of VEEV‐infected neuroblastoma cell cultures. Both NO• and lipid peroxidation were decreased by melatonin treatment in a time‐dependent manner with an associated reduction in iNOS expression 106. Production of brain and serum nitrite, as well as neural lipid peroxidation products, was increased in VEEV‐infected mice. Melatonin treatment curtailed nitrite concentrations in the brain and serum of infected mice and lowered lipid peroxidation products 107.

Respiratory syncytial virus is a common cause of bronchiolitis, a severe lower respiratory tract affliction that infects nearly all infants by age three worldwide. Mice inoculated intranasal with RSV showed elevated oxidative stress due to rises in NO• and •OH. Also elevated malondialdehyde (MDA) and decreases in GSH and SOD activities were observed. Pre‐administration of melatonin in vivo resulted in marked reduction of acute lung oxidative injury induced by RSV, suppressed MDA, NO• and •OH generation, and restored GSH and SOD levels in the lungs of RSV‐infected mice 31.

Rabbit hemorrhagic disease virus (RHDV) causes bleeding in the respiratory system, liver, spleen, cardiac muscle,and occasionally in the kidneys of infected rabbits with mortality over 90% in adults 108. The activity and mRNA expression of the antioxidants enzymes GPx, glutathione‐s‐transferase (GST) and Mn‐SOD were significantly reduced in the liver of RHDV‐infected rabbits used as a model of fulminant hepatic failure; these changes were reduced by melatonin administration in a concentration‐dependent manner. Melatonin treatment also caused a rise in protein expression of the nuclear factor erythroid 2 (Nrf2), a transcription factor that plays a critical role by binding to the antioxidant response element in the promoter region of a number of genes encoding for antioxidant and detoxifying enzymes in several types of cells and tissues 109. The activation of Nrf2 during prevention of oxidative liver injury by melatonin in rats treated with dimethylnitrosamine has been reported 110.

MELATONIN AS A POSITIVE REGULATOR OF IMMUNE FUNCTIONS IN VIRAL INFECTIONS

During the early phase of infection and depending on the nature of the infected cells and the infecting virus, early innate defense mechanisms may be triggered to limit the extent of viral spread. The first mechanism to limit the extent of viral spread is the recognition of pathogen‐associated molecular patterns (PAMPs), which are mostly viral nucleic acids, or their synthetic analogs produced during the viral infection, by a large repertoire of pattern recognition receptors (PRRs), including Toll‐like receptors (TLRs), Nod‐like receptors (NLRs), RIG‐I‐like receptors (RLRs) and AIM2‐like receptors (ALRs) 111, 112, 113, 114. Such recognition initiates signaling cascades that culminate in the activation of transcription factors including nuclear factor kappa B (NF‐κB), activating transcription factor 2 (ATF‐2), activating protein‐1 (AP‐1) and interferon regulatory factors 3 (IRF3) and 7 (IRF7). These stimulate the expression of type I IFN genes that are synthesized in most cell types and especially in plasmacytoid dendritic cells (pDC) 115. All IFNs bind to specific ubiquitously expressed cell surface receptors and induce a large number of interferon‐stimulated genes (ISG), whose encoded proteins mediate the antiviral effects of interferons.

Among these ISGs, dsRNA‐activated protein kinase (PKR) primarily inhibits replication of RNA viruses such as vesicular stomatitis virus (VSV), EMCV, WNV, HCV and DNA viruses including HSV‐1 116. Another group of ISGs is the 20–50‐oligoadenylate synthetases (OAS) that requires dsRNA for its activation and is a major antiviral effector against picornaviruses (e.g. EMCV) and influenza A virus, as well as other RNA viruses 117. Non‐specific ssRNA cleavage also occurs after induction of ISG20, a 30‐exoribonuclease, which contributes to inhibition of RNA viruses such as VSV 118. An additional, non‐enzymatic mechanism of translation inhibition is pursued by the ISG56/IFIT family proteins, which act against HCV 119, 120, 121. Another IFN‐induced protein is the human MxA, which is a key component in innate defense against orthomyxoviruses such as influenza virus as well as measles virus, VSV, Hanta virus and SFV 116, 122, the Viperin (CIG5), which might interfere with viral budding of enveloped viruses, such as CMV, HCV, and influenza virus 123, and the nucleic acid‐editing enzymes APOBEC3G and −3 F, which inhibit retroviruses 124.

A second mechanism is the triggering of effector functions of cellular components of the innate immune system, such as granulocytes, natural killer cells (NK) and natural killer T cells (NKT cells), macrophages, and dendritic cells, which are normally rapidly recruited and/or activated at the site of virus infection, causing a local inflammation 125. During this early phase, activated NK cells release IFN‐γ, which is not stimulated by viral PAMPs but by IL‐12 and IL‐18 released by activated macrophages 126. All of the cellular components of the innate immune system can participate in the antiviral response by killing infected cells, by producing chemokines (including eotaxin, RANTES, MCP‐1, IL‐8) that recruit inflammatory cells into the infected tissue and by producing antiviral and immunoregulatory cytokines (including TNF‐α, IL‐1, IL‐3, IL‐4, IL‐5, IL‐6, IL‐12, IL‐18, GM‐CSF) that enable the adaptive immune response to recognize infected cells and perform antiviral effector functions 127, 128, 129, 130. Lymphocytes are cells of this adaptive immune system. Among them, two subsets of CD4+ T cells, Th1 and Th2, play a key role in antiviral immunity. After being stimulated by antigen presenting cells, Th1 cells produce IL‐2, TNF‐α and IFN‐γ, which possess antiviral activities and regulate activation of CD8+ cytotoxic T cells, whereas Th2 cells produce IL‐4, IL‐5, IL‐10 and IL‐13, which stimulate B cells to produce antibodies 131. Despite the fact that virus‐specific Th2 cells can be detected following primary infection by any virus, virus‐specific Th1 cells are usually much more abundant and reach very high numbers at the peak of the acute infection 132. Moreover, their frequencies remain elevated following resolution of the infection.

Melatonin is synthesized in lymphoid organs, such as the bone marrow, thymus and lymphocytes 133, 134, 135, and there are high affinity membrane melatonin receptors as well as nuclear binding sites in circulating lymphocytes, spleen cells and thymocytes 136, 137, 138. Melatonin is known to activate both innate and adaptive immune responses leading to an increase in immune responsiveness and regulation of several immune functions 27, 28, 139, 140, 141, 142, 143. Melatonin has properties as an inflammatory regulator, because it differentially modulates pro‐inflammatory enzymes, and controls the production of inflammatory mediators such as cytokines and leukotrienes. The timing of its pro‐inflammatory and anti‐inflammatory effects suggests that melatonin might promote early phases of inflammation, on the one hand, and contribute to its attenuation on the other hand, to avoid complications of chronic inflammation 144. Melatonin enhances the production of IL‐1, IL‐6, TNF‐α and IL‐12 from the monocytes 145 and of IL‐2, IFN‐γ and IL‐6 from cultured human peripheral blood mononuclear cells 137. It has been suggested that melatonin and IFN‐γ create an immunoregulatory circuit responsible for the antiviral, antiproliferative and immunomodulatory actions of IFN‐γ 146. This cytokine increases serotonin and melatonin levels in lymphocytes and macrophages. The early stimulation in the production of IFN‐γ by melatonin suggests that earlier treatment with this indoleamine could increase the antiviral activity of IFN‐γ 147. In addition to stimulating the production of several cytokines that regulate immune function, melatonin enhances immune function by directly stimulating polymorphonuclear cells, macrophages, NK cells and lymphocytes 148. Recently, considerable attention has been focused on the fact that melatonin treatment has been found to augment CD4+ T cells in lymph nodes of rats 149. Consequently, melatonin is considered an immunoenhancing agent 141, 150.

In retrovirus‐infected people and mice, whereas Th1 cytokine (IL‐2 and IFN‐γ) production declines, Th2 cytokine (IL‐4, IL‐5, IL‐6, and IL‐10) production increases 151, 152, 153. The excessive Th2 cytokines suppress Th1 cells, causing anergy of cell‐mediated immunity, thus allowing the retrovirus as well as normal flora to reproduce and promote free radical generation by macrophages 154. Female C57BL/6 mice infected with the LP‐BM5 MLV develop murine AIDS. Treatment with melatonin, alone or with dehydroepiandrosterone (DHEA), prevented retrovirus‐induced reduction in B‐cell and T‐cell proliferation and in Th1 cytokine secretion, as well as overproduction of Th2 cytokines and TNF‐α 155. In fact, melatonin alters the balance of Th1 and Th2 cells mainly towards Th1 responses increasing the production of Th1 cytokines 156.

A link between melatonin and the immune system has been also reported in patients infected with HIV‐1. Although mean serum IL‐12 levels in HIV‐1‐affected individuals did not significantly differ from healthy controls, the IL‐12 levels of HIV‐1 patients with advanced disease (CDC stage C) were significantly lower than those of patients in less advanced CDC stages B and A. Taking into account that serum IL‐12 levels run parallel with serum melatonin concentrations as the disease advances, a relationship between immune function and melatonin has been suggested; a reduction in serum melatonin could possibly affect IL‐12 production thereby contributing to the progress of HIV‐1 infection 157.

The protective effect of melatonin against VEEV by regulation of the immune system has been described by Bonilla et al. 158. The endogenous production of IFN‐γ, IL‐1β and TNF‐α, but not of IL‐2 and IL‐4, is stimulated in VEEV‐infected mice treated with melatonin 159. Nevertheless, the average mortality obtained during neutralization experiments with the corresponding anticytokine antibody suggests that although neither TNF‐α nor IFN‐γ is essential for the protective effect of melatonin observed in murine VEEV infection, IL‐1β induced by melatonin treatment is a target cytokine to promote the immune enhanced state. This in turn causes the viral clearance or helps generate an earlier immune response against the VEEV infection 160. In contrast, in the brain of VEEV‐infected mice, melatonin stimulates the endogenous production of IL‐1β but reduces the concentration of TNF‐α 158. IL‐1β is considered one of the earliest host mediators during infectious diseases of the CNS and its role in infectious processes of the brain parallels its role in the peripheral immune system 160. Although IL‐1β deficiency is protective against fatal Sindbis virus infection 161, mice deficient in IL‐1β have increased susceptibility to influenza virus 162. In poxvirus animal models, the viral induction of this cytokine is also beneficial for the host 163. The increase in IL‐1β levels detected in blood and in brain of VEEV‐infected mice after melatonin treatment also plays a protective role, possibly by neuronal support and protection by inducing nerve growth factor secretion by astrocytes 164. This supplies a trophic factor for many neuronal cell types in times of stress such as that produced by VEEV infection.

The significant reduction in the concentration of brain TNF‐α induced by melatonin in VEEV‐infected mice likely diminishes the inflammatory response caused by the migration of granulocytes and macrophages to inflammatory sites within the CNS 165. These cells are recruited by colony‐stimulating factors produced by astrocytes stimulated by TNF‐α and as a consequence of alterations in blood–brain barrier (BBB) permeability caused by the adhesive properties of astrocytes stimulated by TNF‐α. TNF‐α is known to induce intercellular adhesion molecules on neighboring endothelial cells 166, alter BBB permeability and promote inflammatory cell infiltration into the CNS. By reducing adhesion molecule production, which melatonin is known to do 167, the indole would protect the brain infected with VEEV.

Respiratory syncytial virus bronchiolitis in infants is characterized by a massive infiltration of inflammatory cells into the airways. Of the diverse intracellular signaling pathways, RSV is recognized by TLR3, which initiates a signaling cascade that culminates in the activation of the transcription factor NF‐κB; NF‐κB is a central mediator of RSV‐induced airway inflammation in vivo 145, 168, 169. RSV infection of RAW264.7 macrophages time‐dependently stimulates the rapid activation of TLR3 and NF‐κB, as well as subsequent NF‐κB dependent genes, many of which encode for pro‐inflammatory cytokines and chemokines including TNF‐α and IL‐1β. Melatonin decreases TLR3‐mediated downstream gene expression in RSV‐infected macrophages in a dose‐dependent and time‐dependent manner. Such inhibition of NF‐κB activity, as well as of TNF‐α in serum, seems to be the key event required to explain the reduction in inflammatory gene expression caused by melatonin 31, 170.

MELATONIN AS A REGULATOR OF PROGRAMMED CELL DEATH IN VIRAL INFECTIONS

As obligate intracellular parasites, viruses are dependent on the host for each stage of replication and, therefore, constantly interface with multiple components of the host cell machinery, including cellular receptors and uptake pathways, gene expression mechanisms and the cell division apparatus. Viral utilization of these systems likely causes cell stress and activates death‐signaling pathways or alters expression of genes that control cell survival, evoking PCD 171, 172.

Apoptosis is one type of PCD, which is dependent on cleavage of important cellular factors by effector caspases such as caspase‐3 and caspase‐7. Two major pathways govern the activation of such effector caspases. In the intrinsic pathway, intracellular stresses sensed by the BH3‐only members of the bcl‐2 family promote the formation of the apoptosome by activation of caspase‐9 through release of proapoptotic molecules such as cytochrome c and Smac/Diablo from the mitochondria. The apoptosome directly activates effector caspases. In the extrinsic pathway, occupation of death receptors such as Fas and tumor necrosis factor receptor (TNF‐R) by death ligands including FasL and TNFα forms a death‐inducing signaling complex (DISC). This results in the activation of the initiator caspase, caspase‐8, which directly mediates effector caspase activation and causes cell death.

The ability of melatonin to modulate apoptosis and to differentially regulate the expression of pro‐apoptotic and anti‐apoptotic mediators has been reported in many studies 29, 173, 174, 175, 176, 177. RHDV infection induces liver apoptosis with increased caspase‐3 expression and activity 178, 179. These effects are attenuated by melatonin in a concentration‐dependent manner. Anti‐apoptotic actions of melatonin on the intrinsic pathway were related to a reduced expression of Bax and cytosolic cytochrome c release, increased expression of Bcl‐2 and Bcl‐xL, and inhibition of caspase‐9 activity. Melatonin treatment also has effects on extrinsic pathway resulting in a reduction in caspase‐8 activity, TNF‐R1 expression and phosphorylated Janus kinase (JNK) expression, and increased expression of cellular FLICE‐inhibitory protein (c‐FLIP), an inhibitor of caspase‐8 179. These findings show that inhibition of apoptotic mechanisms contributes to the beneficial effects of melatonin in rabbits with experimental infection by RHDV and supports a potential hepatoprotective role of melatonin in fulminated hepatic failure.

Autophagy is a type of PCD characterized by the formation of autophagosomes to remove excessive proteins and thereby maintains homeostasis within the cell. Autophagy is now recognized as a component of both innate and adaptive immune responses to bacterial and viral pathogens 180. Varicella zoster virus infection provides an excellent example of autophagy in humans, because abundant autophagosomes are easily detected in the skin vesicles of both varicella and zoster 181. Autophagy is also found during viral replication of HCV 182, rabbit calicivirus 183 and poliovirus 184. Given that melatonin modulates autophagy through redox‐sensitive transcription factors 185, the role of melatonin in such viral infections involving autophagy should be examined.

MELATONIN AS A CO‐TREATMENT IN VIRAL INFECTIONS

Beneficial effects of melatonin when combined with several drugs, such as doxorubicin, cisplatin, epirubicin, cytarabine, bleomycin, gentamicin, cyclosporin, indometacin, acetylsalicylic acid, ranitidine, omeprazole, isoniazid, iron and erythropoietin, phenobarbital, carbamazepine, haloperidol, caposide‐50, morphine, cyclophosphamide and L‐cysteine have been reported 24. Recently, a single blind randomized study showed a higher percent of a complete regression of symptoms of HSV‐1 infection after a treatment with melatonin plus SB‐73 (an extract of Aspergillus sp. with anti‐herpetic properties) compared with the treatment with acyclovir alone 186. Effects of melatonin to increase the efficacy of other antivirals should be studied.

CONCLUDING REMARKS AND PERSPECTIVES

Melatonin is an endogenously produced and ubiquitously acting molecule 187, 188, 189. Because of its highly diverse actions, this indoleamine has potential to combat a wide variety of pathophysiological conditions 190, 191, 192, 193, 194; it has been tested in numerous clinical trials 23 with the outcomes of the treatments always being beneficial. Because of its essential and basic actions on cell physiology, melatonin qualifies for the moniker “molecular handyman,” as indicated in the title of this review.

In relation to viral infections, melatonin also seems to be beneficial as indicated in the experimental studies summarized herein. Its favorable actions against viral infections likely relate to its ability to limit the negative molecular processes normally activated when viruses invade cells. Melatonin's actions include an ability to promote immune surveillance, to scavenge free radicals thereby significantly reducing the associated molecular destruction and to modulate the processes related to apoptosis. These multiple actions suggest that melatonin should be evaluated in randomized controlled trials as a preventive agent or as a treatment of viral infections particularly in older individuals where endogenous levels of melatonin have declined. It is the hope of the authors that this summary will stimulate interest in experimental examination of melatonin's antiviral actions.

CONFLICT OF INTEREST

The authors have no competing interest.