1 INTRODUCTION

Obesity is a public health problem with pandemic proportions, which has prompted the scientific community to better study the molecular mechanisms that can be effectively targeted by therapeutic strategies.1 Obesity is characterized by a significant increase in adipose tissue caused by an imbalance in food intake and energy expenditure. It is a pathological condition that includes multifactorial aspects, including genetic and endocrine defects.1 Moreover, obesity represents a crucial health problem both in developed and developing countries that is mainly linked to a general change in human lifestyle, associated with a sedentary lifestyle and availability of high-energy refined foods.1, 2 The failure of the healthcare system in treating obesity during its early stages makes the management of patients with obesity a greater challenge because it requires the concomitant presence of several factors. These factors include the psychological predisposition of patients to ameliorate their physical illness and a multidisciplinary medical approach that can overload the healthcare system and dramatically impact economic and social costs.1, 3, 4

Obesity has long been associated with increased morbidity and mortality caused by cardiovascular disease,5 diabetes,6 hyperlipidemia,7 and complications from and predisposition to infectious diseases.8 Complex modifications to adipose tissue arrangement, including immune cell infiltration and pro-inflammatory cytokine secretion, impact predisposition to several infectious diseases.8 Obesity is closely related to chronodisruption, which is characterized by the deregulation of physiological and behavioral central and peripheral circadian rhythms that contribute to obesity-related metabolic impairment. Eating and sleeping time schedules can strongly influence the hunger-satiety circuit and body weight gain, which are relevant synchronizers of the human biological clock9 and in which melatonin plays an important role. Moreover, its intake has been suggested as a potential regulator of circadian rhythms,10 the molecular mechanisms of which underlie the onset of obesity,11, 12 innate and adaptive immune responses,13 and oxidative reactions during infections.14-17

Here, we present a comprehensive review that discusses the main molecular mechanisms triggered by melatonin in the control of infections in an obesity setting, focusing on the regulation of chronic low-grade inflammation (metaflammation) that occurs primarily through the antioxidant properties of melatonin and its ability to entrain the central and peripheral circadian rhythms.

2 SEARCH STRATEGY

The current review critically analyzes the basic and translational evidence of the role of melatonin in the regulation of infections during obesity by describing the available literature from 2000 to 2021. Particular attention has been given to the molecular mechanisms implicated in metaflammation and in the regulation of oxidative stress, of which melatonin is a strong modulator. The relevant literature was screened from PubMed (MEDLINE) using the search terms “obesity,” “inflammation,” “metaflammation,” “infections,” “circadian rhythm,” “melatonin,” “melatonin and circadian rhythm,” “melatonin and inflammation,” “melatonin and immune system,” “melatonin and infections,” “melatonin and glucose metabolism,” “melatonin and lipid metabolism,” “melatonin and aging,” “melatonin and thymus involution,” “melatonin and adipogenesis,” “melatonin and bone marrow adipogenesis,” and “melatonin and chemotherapy.”

3 THE LINK BETWEEN OBESITY, METAFLAMMATION, AND INFECTION

Adipose tissue is a metabolically active organ that is tightly interconnected with the immune system. Inflammation plays a pivotal role in the interaction between adipose tissue and the immune system.18 Changes in adipose tissue architecture, including adipocyte hypertrophy and hypoxia and typical features of obesity, can induce pro-inflammatory cytokine production that attracts immune cells at the local level in adipose tissue and triggers chronic low-grade inflammation.19 This chronic state of inflammation is mainly mediated by macrophages whose polarization from the anti-inflammatory (M2) to pro-inflammatory (M1) phenotype is important for the pathogenesis of metabolic disease.19, 20 Along with macrophages, lymphocytes (B and T cells) can infiltrate the hypertrophic adipose tissue and contribute to the increased production and release of pro-inflammatory cytokines, including interleukin (IL)-8, IL-6, and tumor necrosis factor α (TNFα).21 Studies on adipose tissue during obesity onset demonstrated that approximately two-thirds of the stromal vascular fraction in adipose tissue is represented by immune cells, increasing the ability of adipose tissue to act as an immunological tissue and control systemic inflammation and metabolism.18, 22 In turn, the immune system may actively participate in several obesity-linked disorders such as atherosclerosis and nonalcoholic steatosis.22 More recently, the onset of infections has been found to be among the systemic complications associated with obesity.23 Indeed, strong evidence indicates that patients with obesity with systemic metaflammation more frequently experience infections, contract more severe infections, and have poorer prognoses.8

4 MELATONIN: SYNTHESIS AND GENERAL MECHANISMS OF ACTION

Melatonin, or 5-methoxy-N-acetyltryptamine, is an indolamine secreted by the pineal gland during the dark phase of the circadian rhythm in both diurnal and nocturnal species, earning it the nickname “hormone of darkness.”24 Melatonin has a half-life of approximately 30 min and is produced from serotonin through two enzymatic steps: acetylation and transfer of a methyl group. Hence, serotonin and melatonin show opposite circadian secretions.24, 25 The main functions of melatonin are related to circadian rhythms and sleep–wake cycle control.24 Moreover, melatonin is related to a wide range of physiological activities, such as memory, mood, anxiety control, blood pressure reduction, and osteoblast differentiation.24, 26 In addition, melatonin can regulate the secretion of gonadotrophin-releasing hormone from the hypothalamic neurons, stimulate the secretion of progesterone from granulosa cells, and suppress the expression of estrogen receptors,27 thus influencing the reproductive state. Exogenous melatonin is usually administered to treat disturbances of the sleep–wake cycle related to circadian rhythm disruption, such as jet lag,28 and it has recently emerged as a supplement to conventional therapies for several diseases, including cancer.29 The increasing use of melatonin in clinical practice is linked to its powerful anti-inflammatory and analgesic effects.30 All these processes are mediated by the specific binding of melatonin to G protein-coupled receptors that are widely expressed in the central nervous system (CNS) and different peripheral organs; these receptors are divided into two types: melatonin receptor type 1a (MT1) and melatonin receptor type 1b (MT2).24, 27, 31 MT1 is generally expressed in the skin and related to the inhibition of cyclic adenosine monophosphate (cAMP) formation, together with the inhibition of protein kinase A (PKA) and CREB phosphorylation.24, 27, 31 MT1 can also increase the phosphorylation of mitogen-activated protein kinase 1/2 (MAPK1/2), including the extracellular signal-regulated kinase 1/2 (ERK1/2), as well as potassium conductance. Similarly, activation of the MT2 receptor leads to inhibition of both cAMP and cGMP formation, activation of protein kinase C (PKC) in the suprachiasmatic nuclei (SCN), and decreased calcium-dependent dopamine release in the retina.24, 27, 31

The activation of MT1 receptors leads to vasoconstriction, indicating that vascular tone is differently regulated by melatonin receptors, whereas the activation of MT2 receptors leads to vasodilation.24, 27, 31 Moreover, melatonin interacts with intracellular proteins, such as calmodulin, calreticulin, and tubulin, suggesting melatonin may act as an antiproliferative agent in cancer.32 Regarding immunomodulatory effects of melatonin, the expression and function of the retinoid-related orphan nuclear hormone receptor (RZR/ROR) family was recently shown to be modulated by melatonin.33 It has been demonstrated that IL-2 and IL-6, which are the main interleukins involved in immune and inflammatory responses, are produced following the activation of nuclear melatonin receptor RZR/ROR by human peripheral blood mononuclear cells (PBMCs).34 Moreover, melatonin has also been widely used as an antioxidative agent and free radical scavenger because of its ability to stimulate several antioxidant enzymes, such as superoxide dismutase, glutathione peroxidase, and glutathione reductase.15 The antioxidant properties of melatonin are potentially useful in the prevention of inflammatory diseases, such as liver injury,35 pulmonary,36 cardiovascular diseases,37 and cancer.38

5 THE ROLE OF MELATONIN IN OBESITY 5.1 Melatonin: Implications of chronobiology on metabolic pathways

Melatonin is well known as one of the main mediators through which the central master clock coordinates several areas of the CNS and the peripheral organs, acting as an internal synchronizer, also named “internal zeitgeber”24 (Figure 1). Melatonin production from the pineal gland is strictly controlled by the circadian rhythm and is chemically expressed in the dark. For this reason, melatonin production is always circumscribed to the night, regardless of the behavioral distribution of activity and the rest of mammalian species (diurnal, nocturnal, or crepuscular species).24 In humans, the endogenous circadian clock regulates a wide range of physiological activities and is formed by a master clock residing in the suprachiasmatic nuclei of the hypothalamus, which receives information from external and internal inputs to synchronize the peripheral clocks with these inputs.39, 40 Master and peripheral clocks are composed of the same clock genes and proteins that oscillate at the single-cell level and are finely regulated by transcriptional and translational feedback loops.41 Specifically, two transcriptional activators, BMAL1 (brain and muscle aryl hydrocarbon receptor nuclear translocator [ARNT]-like protein 1) and CLOCK (clock circadian regulator) form the BMAL1/CLOCK complex by heterodimerization in the cytoplasm that then translocates into the nucleus where it induces the gene expression of PERIOD (PER1/2/3) and CRYPTHOCROME (CRY1/2).42 PER and CRY are two gene transcription factors for the PER/CRY complex by heterodimerization in the cytoplasm and also translocate into the nucleus to repress gene expression and the activity of the BMAL1/CLOCK complex, which is also able to repress PER/CRY protein expression and activity through proteasome-mediated pathway activation.42 In this way, the BMAL1/CLOCK and PER/CRY complexes make a negative feedback loop to control each other's function. Clock genes and proteins are rhythmically expressed during the day and cooperate to induce clock machinery function.42 As major regulators of the master and peripheral clock, BMAL1/CLOCK can modulate the expression of several genes dislocated in various tissues,43 mainly in the mouse liver, where Bmal1 has been demonstrated to bind 2000 target genes in a circadian-dependent manner, with a peak of DNA binding occurring during the day.44 Chromatin immunoprecipitation sequencing (ChIP-seq) has been used to analyze mouse hepatic gene binding sites for clock genes, and we confirmed that Bmal1 binds genes during the middle of the day, whereas gene ontology analysis revealed a large predominance of gene expression regulated by Bmal1, particularly genes involved in metabolic pathways.45 Moreover, the maximum occupancy of transcriptional activators by Bmal1/Clock genes occurs concomitantly with H3K9 acetylation, which is characteristic of an active promoter region.45 In addition, in mouse intestine tissues, Bmal1 can regulate the circadian expression of three peroxisome proliferator-activated receptor (PPAR) target lipid enzymes: acyl-CoA oxidase (Aox), 3-hydroxy-3-methylglutaryl coenzyme A (Hmg-CoA) synthase and cellular retinol-binding protein II (CrbpII) by directly acting on the promoter activities of these genes.46 Conversely, Per and Cry bind mouse hepatic genes during the night concomitantly with Hk34 trimethylation, characteristic of an inactive promoter region.45 Moreover, Per2-deficient mice have been reported to show a strong reduction in total triacylglycerol and nonesterified fatty acids, resulting in dysregulated lipid metabolism. Per2 can inhibit PPARγ recruitment to target promoters, resulting in transcriptional activation.47

Melatonin regulation of peripheral clocks. All vertebrates synthesize and release melatonin through the pineal gland in a circadian-dependent manner that has a classical peak during the night and is suppressed during the day. The nocturnal synthesis and release of melatonin are highly controlled by the master clock residing in the suprachiasmatic nucleus (SCN) through a fine molecular mechanism that is inhibited by light exposure. In turn, melatonin synchronizes peripheral clocks by acting at different levels and regulating several physiological functions. When melatonin synthesis and secretion are deregulated and/or desynchronized due to loss of master clock function, there is misalignment of peripheral clocks and a failure of physiological function control. Illustrations created with BioRender.com

Melatonin induces phase shifts and entrainment of the circadian clock by modulating clock gene expression and neuroplasticity.24, 48, 49 Thereafter, nocturnal melatonin not only conveys temporal information to peripheral clocks in several organs but also harmonizes the central and peripheral clocks, improving circadian coordination.50

Along with its synchronizing ability, melatonin is a key mediator between the circadian rhythm and lipid and carbohydrate metabolism at different tissue levels, such as skeletal muscle, adipose tissue, and the liver (Figure 2).

Molecular mechanisms induced by melatonin in different peripheral targets. Melatonin entrains the expression of clock genes in hepatocytes, reduces lipid accumulation, and decreases hepatic steatosis by downregulating Fas, Pparγ, and Srebp1 gene expression in the liver. The action of melatonin on white adipocytes induces a switch to the brown adipocyte phenotype, activating thermogenic action through stimulation of Ucp-1, Irisin, and Pparγ gene expression. The lack of melatonin action in muscle cells induces a loss of the physiological rhythm of glucose. Illustrations created with BioRender.com

In particular, the effect of circadian rhythm disruption in skeletal muscle has been linked to dysregulation of glucose internalization,51 insulin signaling,52 and physical training.53 MT1 and MT2 receptor knockout (KO) mice showed conserved Per2 and Bmal1 circadian expression but a loss of the blood glucose physiological rhythm.54 Circadian rhythms play a fundamental role in lipid homeostasis, as reported in 10 healthy subjects with physiological cortisol and melatonin circulation levels consisting in cortisol nadir at 12:00 pm and melatonin zenith at 3:00 am. Cultured and synchronized myocytes with forskolin from the same subjects showed a superimposed circadian expression of lipid regulated by clock machinery and a diurnal lipid profile that was correlated with the transcription profiles of lipid key enzymes.55 Moreover, Clock mutant mice retained melatonin diurnal oscillation but lost peripheral clock machinery components, particularly Per2, and had impaired glucose tolerance; however, these mice also had reduced plasma free fatty acids, increased plasma adiponectin, and improved insulin sensitivity.56 Analysis of the main enzymes involved in lipid metabolism in epigonadal fat such as hormone-sensitive lipase (hsl), adiponutrin, and desnutrin genes revealed that there was a difference in HSL expression in Clock mutant mice, as well as a loss of Per2 and a conserved Bmal1 genes expression rhythmicity. Conversely, adiponectin and desnutrin expression levels were maintained between mutant and wild type, but the circadian expression showed an anti-phasic trend from 10:00 pm to 8:00 am.56 Moreover, melatonin is synthesized and released during the dark hours of the night together with higher lipid adsorption,57 suggesting a pivotal role of melatonin and the circadian system for the regulation of lipid homeostasis.

Patients with melatonin deficiency due to radiotherapy or surgical removal of the pineal gland who were administered 3 mg melatonin for 3 months displayed improved cholesterol and triglyceride blood levels, although body weight and liver fat remained unchanged.58 These patients showed increased brown adipose tissue volume and activity, indicated by positron emission tomography–magnetic resonance imaging (MRI) measurement.58 In animal models, melatonin was also reported to be important for the regulation of lipid levels and body fat composition. Male mice fed a high-fat diet (HFD) and exposed to constant light for 24 h showed increased body weight and insulin resistance onset than mice in the control group, who were exposed for 12 h dark and 12 h light.59 Moreover, the same mouse model exposed to constant light exhibited increased absorption and digestion of lipids. In addition, male obese, high fat and high caloric diet-induced rats treated for 8 weeks with 10, 20, and 50 mg/kg body weight melatonin showed a significant increase in circulating irisin, a myokine that is particularly important in the switching of white adipose tissue into brown adipose tissue.60 Furthermore, these doses of melatonin significantly reduced white adipocyte levels in inguinal depots and in the same district, as well as significantly increased Pparγ and uncoupling protein-1 (Ucp-1) gene expression levels that are associated with decreased lipoprotein (Lpl) transcriptional levels, thus confirming the browning transformation of white adipose tissue at the morphological and molecular level.60 Similarly, male pinealectomized rats supplemented with melatonin showed higher physiological UCP-1 gene and protein expression and Hsl gene expression levels in brown adipose tissue than in non-treated rats.61 Moreover, rat62 and human63 cultured adipocytes express MTs, and 1 nM melatonin was reported to regulate lipolysis isoproterenol-induced when administered for 4 h.62 Moreover, 1 nM melatonin combined with 5 nM insulin for 6 h is able to stimulate leptin secretion and gene expression f in cultured rat adipocytes.64

The role of melatonin in the regulation of pluripotent mesenchymal stem cells (MSCs) in the bone marrow and adipose tissue has emerged. Human MSCs cultured in adipogenic-inducing medium containing 10−4 M melatonin showed the downregulation of mRNA expression levels of markers of terminal adipocyte differentiation, such as LPL, adiponectin, and leptin and the upregulation of osteoblast differentiation markers, suggesting both anti-adipogenic and anti-osteoporotic effects.65 Interestingly, the protective role of melatonin against bone marrow damage, particularly cadmium-induced damage, has been demonstrated in vitro.66 Human adipose MSCs chronically exposed to cadmium toxicity (e.g., 21 days of treatment with 0.5–2 μmol/L cadmium) showed a decrease in cellular lipid accumulation and attenuation of osteogenic response when cells were exposed to 50 nmol/L melatonin for 4 h compared with cells treated with cadmium and melatonin alone, respectively.66 These results confirmed that melatonin inhibited the adipogenic differentiation of MSCs induced by cadmium exposure, leading to a preferential commitment of the precursor cell toward an osteogenic lineage. Similarly, the involvement of AMPK/β-catenin signaling has been reported in C3H/10T1/2 mouse pluripotent stem cells.67 Exposure to 100 μM melatonin for 3, 5, and 7 days promoted the osteogenic differentiation of C3H/10T1/2 cells and showed a synergistic effect when combined with bone morphogenetic protein 9 (BMP9). This effect was mediated by the phosphorylation of Smad1/5/8 and β-catenin and by the activation of AMPKα proteins, which are the main factors involved in osteogenic cellular lineage development.67

The anti-lipogenic effect of melatonin in patients with nonalcoholic fatty liver disease (NAFLD), as well as the molecular mechanisms involved, such as the scavenger activity via stimulation of antioxidative enzymes has been investigated in in vivo human and animal models.68-71 Conversely, a recent study comparing clock gene expression in PBMCs before and after a period of alcohol consumption in daytime and nighttime workers demonstrated significant differences in melatonin secretion and clock gene expression.72 In the nighttime workers, alcohol consumption delayed physiological melatonin secretion, increased the total amount of PER1 expression, and decreased the total amount of CRY1 expression compared with daytime workers.72 Similar results have been reported in obese mice exposed to constant light, fed with HFD and supplemented with 50 mg/kg body weight melatonin from 8:00 pm to 8:00 am.59 After 10 weeks of melatonin exposure, obese mice showed decreased liver weight and hepatic lipid content levels, decreased circulating levels of glycemia, and ameliorated insulin sensitivity when compared with animals that did not receive melatonin treatment used as control group.59

As demonstrated at the molecular level, constant light exposure can cause an anti-circadian expression of clock genes in mouse liver tissues that restore the physiological pattern with melatonin supplementation.59 The main proteins involved in cellular and tissue lipid accumulation, such as fatty acid synthase (FAS), cluster of differentiation 36 (CD36), PPARγ, and sterol regulatory element-binding transcription factor-1 (SREBP-1), were upregulated in the liver of mice exposed to constant light, whereas they were decreased when the same mice were treated with 50 mg/kg body weight melatonin, suggesting a clear role for melatonin in the regulation of light/dark cycle and, consequently, lipid metabolism.59 Figure 3 illustrates the effects of melatonin on the metabolic profiles and hepatic lipid homeostasis of mice exposed to constant light conditions.

Metabolic and immune effects of melatonin in rodent models. High-fat diet (HFD) mice exposed to constant light for 24 h showed dysregulated circadian rhythms accompanied by increased body weight. On the hepatic level, mice displayed upregulated levels of Fas and Srebp1 genes, which are associated with hepatomegaly and steatosis. These mice were also hyperglycemic and insulin resistant, and treatment with melatonin improved their metabolic profile by decreasing body weight and downregulating Fas and Srebp1 gene expression in the liver. Thus, hepatic steatosis decreased, and systemic insulin sensitivity and glycemia were improved.59 Pinealectomized rats exposed to constant light showed decreased levels of thymosin α1 and thymulin, which are important factors secreted by the thymus involved in the maturation of immune cells (B cells, T cells, and natural killer cells). Interestingly, melatonin supplementation in this rat model promoted the secretion of thymosin α1 and thymulin, thus improving cell-mediated immunity.83 Illustrations created with BioRender.com

Melatonin supplementation improved the gut microbiota composition of HFD-fed mice exposed to constant light by increasing the genera Roseburia and Eubacterium and reducing the ratio between Firmicutes and Bacteroidetes, which are butyrate-producing bacteria.59 Similarly, the anti-obesogenic effects of melatonin on gut microbiota modulation have been demonstrated.73 HFD-fed mice treated daily for 2 weeks with 0.4 mg/ml melatonin showed improved visceral and subcutaneous adipose tissue accumulation when compared with those were not supplemented with melatonin used as control group. Similar to what was demonstrated in the control group, HFD mice supplemented with melatonin showed restored rhythmicity of clock genes that were otherwise lost as a result of HFD.73 Fecal microbiota was explanted from the control, HFD, and HFD melatonin-treated mice groups and transplanted into antibiotic-treated mice at two different time points (8 am and 4 pm) to investigate the circadian response to HFD feeding. Although there was no significant difference detected in the dysmetabolism of HFD mice when melatonin was administered in the daytime or nighttime, a significant improvement in lipid content was revealed after circadian gut microbiota transplantation. In detail, transplantation in the control group at 4:00 pm significantly increased subcutaneous inguinal fat and circulating triglycerides compared with transplantation at 8:00 am. Moreover, transplantation in HFD melatonin-treated mice groups showed decreased inguinal, perirenal, and periuterine fat depots when compared with control and HFD mice groups.73 Moreover, no significant difference was observed in body weight gain after transplantation in the two different time points evaluated, and interestingly, the lipid profile of the receiving group was influenced by the time at which the microbiota were transplanted.73 In particular, microbiota transplanted at 8:00 am in the HFD group increased serum triglyceride, cholesterol, and HDL concentrations in the receiving control group. However, this effect was slightly reversed when the control group received transplants from HFD melatonin-treated mice at 8 am. Conversely, serum triglyceride levels decreased when microbiota from the HFD was transplanted into the receiving group at 4:00 pm.73

These clinical and preclinical findings indicate that the role of melatonin in the control of adipose, muscle, and liver tissue fat accumulation, circulating lipid levels, and adipocyte differentiation is clearly linked to the expression of circadian rhythm components.

5.2 Melatonin: Implications of chronobiology on the immune system

The most important evidence of the relationship between immunity and cirrhosis is the rhythmic expression of Toll-like receptors (TLRs) during the day.74, 75 TLR expression and function may be modulated by melatonin,65-69 creating tight crosstalk between melatonin, circadian rhythm, and the immune system. TLRs are proteins expressed on the surface of many cells and within endosomes that play an important role in pathogen recognition and the consequent activation of the innate immune system.76 In vivo and in vitro studies have demonstrated the circadian expression of TLRs and the effects of melatonin in the regulation of TLR expression, thus demonstrating a strong correlation between circadian rhythm and immunity. In particular, TLR9, which belongs to the group of endosomal receptors of the innate immune system and are capable of recognizing hypomethylated CpG sequences from DNA, showed circadian expression and functionality in in vivo studies in humans74 and mice.75 In humans, the daily variations in TLR9 responsiveness may modulate the efficacy of CpG oligodeoxynucleotide (ODN)-adjuvanted immunization,74 whereas in immunized mice, the maximum TLR9 responsiveness is Per2 expression-related and, for this reason, correlated with maximum expression levels of Per2, which helped develop a stronger adaptive immunity 4 weeks after immunization, confirming the circadian regulation of this innate immune pathway.75 Moreover, peritoneal macrophages isolated from circadian-deficient Per2-mutant mice and challenged with different pathogen-associated molecular patterns (PAMPs) targeting multiple TLRs produced significantly lower amounts of TNF-α and IL-12 than macrophages isolated from wild-type mice.75 Similarly, an in vitro model of adherent splenocytes and splenic macrophages demonstrated that TLR3 protein levels fluctuated over the daily light–dark cycle and that mRNA levels of TLR2 and TLR6 exhibited rhythmic expression.75

The inhibitory effect of melatonin on TLR action has been recently demonstrated in in vivo preclinical studies77, 78 and in vitro models.79-81 In an in vivo animal model of ovarian cancer, it was reported that 200 μg/100 g body weight melatonin reduced TLR2 and TLR4 function by abolishing inflammatory status typical of ovarian cancer via myeloid differentiation factor 88 (MyD88) and TLR-associated activator of interferon (TRIF) inhibition,77 suggesting that melatonin can influence TLR expression by modulating the inflammatory responses observed in cancer. Moreover, a KO TLR2 mouse model demonstrated a feedback loop between TLR activation and melatonin synthesis and action.78 Mice with allergic airway inflammation induced by intraperitoneal injection of ovalbumin showed increased TLR2 expression associated with increased activation of the nucleotide oligomerization domain-like receptor family, pyrin domain containing-3 (NLRP3) inflammasome and airway inflammation, which were significantly reduced in TLR2 KO mice. Additionally, wild-type mice exhibited reduced melatonin biosynthesis, which was otherwise restored in the TLR2 KO mice,78 demonstrating that the TLR2–NLRP3-allergic airway inflammation circuit was responsible for decreased melatonin secretion. Similarly, in vitro experiments with macrophages demonstrated the inhibitory effects of melatonin on TLR3,80 TLR4,79 and TLR981 pro-inflammatory effects through the prevention of the intracellular activation of pathways typically involved in cellular inflammatory responses, such as ERK1/2 and AKT phosphorylation, NF-κB activation, and MyD88 transcription.79-81

In addition to TLR expression, the role of melatonin in the regulation of immunity related to the circadian rhythm has also been studied in animal models.82, 83 In a rat model of arthritis, decreased levels of melatonin were detected, and ornithine decarboxylase activity (an index of lymph cell proliferation), which is a typical feature of inflammation of the submaxillary lymph node and spleen, was significantly increased, specifically with an acrophase in the afternoon or the morning in lymph nodes and spleen, respectively.82 Surprisingly, daily injection of 100 μg melatonin increased the amplitude of ornithine decarboxylase activity, especially in old rats, suggesting not only the circadian role of melatonin in immune function regulation but also age dependency.82 Moreover, pinealectomized rats continuously exposed to light showed decreased levels of thymosin α1 and thymulin proteins, which are secreted from the thymus and enhanced cell-mediated immunity.83 Moreover, exposure to 10 mg/kg body weight melatonin during the day caused an increase in the levels of thymosin α1 and thymulin in pinealectomized rats, demonstrating the role of melatonin in the regulation of immunity related to circadian rhythms.83 Figure 3 summarizes the effects of melatonin on cell-mediated immunity in pinealectomized rats exposed to constant light conditions.

Taken together, several studies have reported a central role for melatonin in the circadian regulation of metabolic and immune functions in human and animal models as well as in in vitro models, suggesting a pivotal therapeutic role for melatonin in the entrainment of metabolism and the immune system.

5.3 Melatonin: Implications of chronobiology on aging and immune system

Melatonin levels gradually decrease over the lifespan,10 and this condition may be related to lowered sleep efficacy and circadian rhythm disruption. Diminished nocturnal melatonin secretion with associated disturbances in the sleep–wake rhythm has been reported in elderly healthy subjects.84 Moreover, an in vivo study performed in a hamster model revealed that the expression of circadian clock machinery components was highly related to age, as demonstrated by the reduction of Bmal1, Clock, and Per1 gene expression in aged hamsters subjected to a light stimulus.85 Moreover, old laboratory mice showed decreased Per2 gene expression in the SNC compared with young adult mice, regardless of light exposure.86 The intricate role of melatonin in the regulation of clock gene expression and sleep–wake cycle modulation has been investigated in mouse models in which age has been correlated with increased daytime sleep and decreased nighttime wakefulness.87 Moreover, old Wistar rats treated with 30 μg/kg body weight melatonin for 11 days showed restored circadian clock gene expression, which was otherwise altered by aging.88 In 24-month-old rats, Bmal1, Cry1, and Cry2 gene expression in the SNC displayed an abolished daily rhythm whose synchronization was restored by melatonin supplementation, suggesting the role of melatonin in the balance of the sleep–wake cycle and circadian rhythm is significantly influenced by age.88 Finally, because melatonin has potent antioxidant and free radical scavenger properties, it may play an important role in all age-related disturbances, including sleep disturbances, thymus involution, and immune system dysfunction.