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Both immune and neurodegenerative mechanisms underlie multiple sclerosis (MS) and its animal model, experimental autoimmune encephalomyelitis (EAE). MS/EAE are triggered by encephalitogenic immune cells, including Th1 and Th17 cells, whereas T regulatory (Treg) cells are involved in inflammation resolution. Pro-inflammatory macrophages/microglia also play a deleterious role in the disease. Seasonal variations in MS relapses, active lesions, and pro- and anti-inflammatory cytokine levels have been described in MS patients and have been related with both perinatal and adult exposure to sunlight and other environmental factors. However, some data in EAE mice suggest that these variations might be, at least partially, endogenously determined. Thus, our objective was to study the effect of the season of birth and disease induction on the course of EAE, and immune cell infiltration in the central nervous system (CNS) in myelin oligodendrocyte glycoprotein (MOG35–55)-induced EAE in 8 weeks old, female C57BL/6N mice maintained under constant, controlled conditions. EAE severity as well as pathogenic (Th1, Th17, macrophages/microglia) and protective (Treg) subsets was found to vary according to the season of birth or of EAE induction. Summer-born or summer-immunized animals developed a milder disease, which coincided with variations in numbers of T effector/regulatory subsets, and significantly low numbers of macrophages/microglia. These results suggest that endogenous rhythms in immune responses might cause seasonal variations in EAE severity, and, maybe, in the course of MS, and that they might be related to macrophages/microglia., The work reported in the present manuscript was supported by the Department of Health of the Andalusian Government (PI-0209-2010, PI-0485-2014, and PC-0019-2017) and the PAIDI Program of the Andalusian Government (CTS160). NA-S was supported by fellowships from the National Net RETICEF for Aging Studies (Red Temática de Investigación Cooperativa en Envejecimiento y Fragilidad; RD06/0013/0001 and RD12/0043/0012; from the Instituto de Salud Carlos III, Spanish Ministry of Science and Innovation) and by a fellowship from the Department of Health of the Andalusian Government (PC-0111-2016-0111), and IC-C was supported by an FPU grant from the Spanish Ministry of Education, Culture and Sport (FPU13/01210).

High-sensitivity C-reactive protein (hsCRP) and homocysteine (Hcy) are inflammation markers but are also related to cardiovascular diseases, disability, or higher risk of death. Although inflammation is considered to be associated with frailty, data regarding the association between hsCRP or Hcy and frailty are controversial or scarce, especially with respect to their association with prefrailty. Thus, our objective was to study the association of hsCRP and Hcy with prefrailty and frailty in 1,211 Spanish men and women aged 65â 98 years from the Toledo Study for Healthy Aging (TSHA) cohort, classified according to Friedâ s criteria. Hcy was independently associated with frailty (odds ratio [OR] = 1.06; 95% confidence interval [CI]: 1.01â 1.12), whereas hsCRP was independently associated with both prefrailty (OR = 1.03; 95% CI: 1.01â 1.06) and frailty (OR = 1.07; 95% CI: 1.02â 1.12). Furthermore, both markers were positively correlated with the number of Friedâ s criteria that were met and were independently associated with the criteria of exhaustion (Hcy: OR = 1.03, 95% CI: 1.00â 1.06), weakness (hsCRP: OR = 1.03, 95% CI: 1.01â 1.05), and low physical activity (hsCRP: OR = 1.04, 95% CI: 1.02â 1.06). Thus, our results highlight the importance of inflammation in age-related physical decline and, in particular, its association with fatigue, low strength, and decreased physical activity., This study was supported by the Instituto de Salud Carlos III (Red Temática de Investigación Cooperativa en Envejecimiento y Fragilidad; RD06/0013/0001, and RD12/0043/0012). Ministerio de Ciencia e Innovación; PI07/90175). NA-S was supported by fellowships from the Instituto de Salud Carlos III (Red Temática de Investigación Cooperativa en Envejecimiento y Fragilidad; RD06/0013/0001 and RD12/0043/0012) and by a fellowship from the Consejería de Salud, Junta de Andalucía (PC-0111-2016-0111). IC-C was supported by fellowships from the Ministerio de Educación, Cultura y Deporte (FPU13/01210) and Junta de Andalucía (CTS160).

Objectives To obtain greater knowledge of the extra-pineal sources of melatonin during development, the amount of indolamine and the expression levels of the last two enzymes involved in its biosynthesis, Arylalkylamine N-acetyltransferase (AANAT) and acetylserotonin O-methyltransferase (ASMT), were analyzed in the human thymus from children from three different age groups (from days to years). The melatonin membrane and nuclear receptor expression levels also were studied. Methods Quantitative reverse transcriptase PCR and western blot were performed to investigate the receptor and enzyme expression levels. The results were examined and correlated with the ages of the thymuses. Results We found high levels of indolamine in the thymuses of newborns (younger than 1 month), which decreased during development; thymuses from the months (from 2 to 11 months) and years (from 1 to 12 years) groups showed lower levels. A similar decline was also observed in the mRNA of the AANAT enzyme and the expression levels of melatonin receptors. However, ASMT expression was exactly the opposite, with low levels in the newborn group and higher levels in the years group. Our results show that the thymic synthesis of melatonin occurs very early in childhood. Additionally, this is the first report that is focused on melatonin receptors expression in the human thymus. Conclusion Considering the limited melatonin synthesis performed by the newborn pineal gland, we suggest that the high levels of melatonin found in human thymus in this experimental group arise from synthesis in the tissue itself, which could be contributing to the immune efficiency at the thymic level., Highlights • Melatonin biosynthetic machinery in the human thymus from children changes with age. • Melatonin content in the human thymus is higher in newborns. • The thymus not only synthesizes melatonin but also responds to it. • The melatonin effector system decreases throughout childhood.

15 Páginas.-- 6 Figuras.-- 1 Tabla, Metabolic-associated fatty liver disease (MAFLD) is the most important cause of liver disease worldwide. It is characterized by the accumulation of fat in the liver and is closely associated with abdominal obesity. In addition, oxidative stress and inflammation are significant features involved in MAFLD. Recently, our group demonstrated that lupin protein hydrolysates (LPHs) had lipid lowering, antioxidant, and anti-inflammatory effects. Sixty male mice fed with a Western diet were intragastrically treated with LPHs (or vehicle) for 12 weeks. Liver and adipose tissue lipid accumulation and hepatic inflammatory and oxidant status were evaluated. A significant decrease in steatosis was observed in LPHs-treated mice, which presented a decreased gene expression of CD36 and LDL-R, crucial markers in MAFLD. In addition, LPHs increased the hepatic total antioxidant capacity and reduced the hepatic inflammatory status. Moreover, LPHs-treated mice showed a significant reduction in abdominal adiposity. This is the first study to show that the supplementation with LPHs markedly ameliorates the generation of the steatotic liver caused by the intake of a Western diet and reduces abdominal obesity in ApoE−/− mice. Future clinical trials should shed light on the effects of LPHs on MAFLD., This research was funded by the Spanish Government, Ministerio de Economía y Competitividad [AGL2012-40247-C02-01 and AGL2012-40247-C02-02], the Andalusian Government Ministry of Health [PC-0111-2016-0111], and the PAIDI Program from the Andalusian Government [CTS160]. G.S.-S. and I.C.-C. were supported by FPU grants from the Spanish Ministerio de Educación, Cultura y Deporte, [FPU16/02339] and [FPU13/01210], respectively. N.Á.-S. was supported by a fellowship from the National Net RETICEF for Aging Studies (RD12/0043/0012 from the Instituto de Salud Carlos III, Spanish Ministerio de Ciencia e Innovación). B.R.-O. was supported by a grant from European Social Fund and Spanish Ministerio de Empleo y Seguridad Social [EJ-086]. A.I.Á.-L. was funded by the Andalusian Government Ministry of Health [PI-0136-2019]. I.B. was supported by the VI Program of Inner Initiative for Research and Transfer of University of Seville (VI PPIT-US).

20 Páginas.-- 2 Figuras.-- 1 Tabla, The effect of GPETAFLR, a peptide isolated from Lupinus angustifolius L. protein hydrolysate (LPH), on osteoclastogenesis was investigated. Human osteoclasts generated from monocytes were used to analyse the effects of GPETAFLR (50–100 µg/mL) on osteoclastogenesis using TRAP reaction, RT-qPCR, and ELISA procedures. LPS enhanced TRAP activity and the expression of osteoclast marker genes (TRAP, OSCAR, RANK, and CATHK) while downregulated the expression of OPG gene in human monocyte-derived osteoclasts. These effects were reduced with GPETAFLR. Moreover, LPS increased the release of osteoclastogenic cytokines (TNF-α, IL-1β, and IL-6) meanwhile GPETAFLR increased the release of anti-osteoclastogenic cytokines (IL-4 and IL-10) in the medium of human monocyte-derived osteoclasts. For the first time, we show that plant peptides from lupine protein hydrolysates have anti-osteoclastogenic activity. These exciting findings open opportunities for developing nutritional strategies with Lupinus angustifolius L. as dietary source of plant proteins, notably GPETAFLR, to prevent development and progression of osteoclast-related diseases., This work was supported by grant AGL2012-40247-C02-01 from the Spanish Ministry of Economy and Competitiveness. Authors thank Cell Biology Unit of Instituto de la Grasa for its assistance during the fulfillment of this work. SMP acknowledges financial support from “V Own Research Plan” (University of Seville).

[Background] Seasonal influenza virus infection is a significant cause of morbimortality in the elderly. However, there is poor vaccine efficacy in this population due to immunosenescence. We aimed to explore several homeostatic parameters in the elderly that could impact influenza vaccine responsiveness., [Methods] Subjects (> 60 years old) who were vaccinated against influenza virus were included, and the vaccine response was measured by a haemagglutination inhibition (HAI) test. At baseline, peripheral CD4 and CD8 T-cells were phenotypically characterized. Thymic function and the levels of different inflammation-related biomarkers, including Lipopolysaccharide Binding Protein (LBP) and anti-cytomegalovirus (CMV) IgG antibodies, were also measured., [Results] Influenza vaccine non-responders showed a tendency of higher frequency of regulatory T-cells (Tregs) before vaccination than responders (1.49 [1.08–1.85] vs. 1.12 [0.94–1.63], respectively, p = 0.061), as well as higher expression of the proliferation marker Ki67 in Tregs and different CD4 and CD8 T-cell maturational subsets. The levels of inflammation-related biomarkers correlated with the frequencies of different proliferating T-cell subsets and with thymic function (e.g., thymic function with D-dimers, r = − 0.442, p = 0.001)., [Conclusions] Age-related homeostatic dysregulation involving the proliferation of CD4 and CD8 T-cell subsets, including Tregs, was related to a limited responsiveness to influenza vaccination and a higher inflammatory status in a cohort of elderly people., This work was supported by grants from the Fondo de Investigación Sanitaria (FIS; PI18/01216), which is co-funded by Fondos Europeos para el Desarrollo Regional (FEDER) and the Junta de Andalucía, Consejería de Economía, Innovación, Ciencia y Empleo (Proyecto de Investigación de Excelencia; CTS2593). The Spanish AIDS Research Network of Excellence also supported this study (RD16/0025/0019). YM.P was supported by the Consejería de Salud y Bienestar Social of Junta de Andalucía through the “Nicolás Monardes” programme (C-0013-2017).

Multiple sclerosis (MS) is a neuroinflammatory disease of the central nervous system in which the immune system plays a central role. In particular, effector populations such as T helper (Th) 1, Th9, Th17, and Th22 cells are involved in disease development, whereas T regulatory cells (Tregs) are associated with the resolution of the disease. Melatonin levels are impaired in patients with MS, and exogenous melatonin ameliorates the disease in MS animal models by modulating the Th1/Th17/Treg responses and also improves quality of life and several symptoms in patients with MS. However, no study has examined melatonin's effect on T cells from relapsing-remitting MS (RR-MS) patients. Therefore, the objectives of the present study were to evaluate the effects of the in vitro administration of melatonin to peripheral blood mononuclear cells (PBMCs) from 64 RR-MS patients and 64 sex- and age-matched healthy subjects on Th1, Th9, Th17, Th22, and Treg responses and to analyze the expression of the melatonin effector/receptor system in these cells. Melatonin decreased Th1 and Th22 responses in patients, whereas it did not affect the Th17 and Treg subsets. Melatonin also promoted skewing toward a more protective cytokine microenvironment, as shown by an increased anti-inflammatory/Th1 ratio. Furthermore, for the first time, we describe the overexpression of the melatonin effector/receptor system in PBMCs from patients with MS; this alteration might be relevant to the disease because acetylserotonin O-methyltransferase expression significantly correlates with disease progression and T effector/regulatory responses in patients. Therefore, our data suggest that melatonin may be an effective treatment for MS.

5 Figuras.-- 2 Tablas, Although cell-free systems and immortalized cell lines have been used to demonstrate the potential health benefits of lupine proteins and peptides, no study has examined the effects of lupine protein hydrolysates (LPHs) on the immune and oxidative responses of non-immortalized human cells. Therefore, the aims of this study were to evaluate the effects of the in vitro administration of LPHs from Lupinus angustifolius on the immunological and oxidative statuses of human peripheral blood mononuclear cells (PBMCs) from 53 healthy donors. LPHs reduced PBMCs proliferation and the levels of Th1, Th9 and Th17 pro-inflammatory cytokines without being cytotoxic. LPHs also skewed the pro-/anti-inflammatory balance towards a Th2 protective response. Additionally, LPHs increased superoxide dismutase and catalase activities, and the total antioxidant capacity (TAC). This study is the first to show that LPHs reduce T cell inflammatory responses and improve the anti-inflammatory/pro-inflammatory cytokine balance and the TAC by PBMCs. Thus, LPHs may represent an effective option for developing nutritional strategies to prevent pathologies with underlying inflammation and oxidative stress., This work was supported by the Spanish Government, Ministerio de Economía y Competitividad [AGL2012-40247-C02-01, AGL2012-40247-C02-02]; the Andalusian Government, Consejería de Salud [PC-0111-2016-0111] and the PAIDI Program from the [CTS160]. IC-C was supported by a FPU grant from the Ministerio de Educación, Cultura y Deporte [FPU13/01210].

Melatonin modulates a wide array of physiological events with pleiotropic effects on the immune system. While the relevance of specific melatonin membrane receptors has been well established for several biological functions, retinoic acid-related orphan receptor alpha (RORα) has been suggested as a mediator of nuclear melatonin signalling by results obtained from pharmacological approaches. However, a melatonin-mediated downstream effect cannot be ruled out, and further evidence is needed to support a direct interaction between melatonin and RORα. Here, we show that RORα is mainly located in human Jurkat T-cell nucleus, and it is co-immunoprecipitated with melatonin. Moreover, immunocytochemistry studies confirmed the co-localization of melatonin and RORα. Melatonin promoted a time-dependent decrease in nuclear RORα levels, suggesting a role in the RORα transcriptional activity. Interestingly, RORα acts as a molecular switch implicated in the mutually exclusive generation of Th17 and Treg cells, both involved in the harm/protection balance of immune conditions such as autoimmunity or acute transplant rejection. Therefore, the identification of melatonin as a natural modulator of RORα gives it a tremendous therapeutic potential for a variety of clinical disorders.

Breakdown in immunological self tolerance, leading to autoimmune diseases such as multiple sclerosis, might arise from immune recognition of self proteins that have undergone heightened posttranslational modification under pathophysiological conditions. A posttranslational modification of particular interest is the deimination of Arg to citrulline, catalyzed by peptidylarginyl deiminase (PAD) enzymes. As a CD4+ T cell-driven model of multiple sclerosis, we used experimental autoimmune encephalomyelitis (EAE) induced with the immunodominant 35–55 peptide of myelin oligodendrocyte glycoprotein (pMOG) in C57BL/6 mice to test whether citrullination of a T cell epitope can contribute to disease etiopathology. Immunization with an altered peptide ligand (APL) of pMOG with an Arg→citrulline conversion at a TCR contact (residue 41) led to the activation of two populations of APL-responsive T cells that either did, or did not cross-react with the native pMOG peptide. This APL could induce EAE. However, this reflected the activation of T cells that cross-reacted with the native pMOG epitope, because prior tolerization of these T cells using pMOG prevented APL-induced EAE. Using a passive transfer model, we found that T cells that responded specifically to the citrullinated form of pMOG were neither necessary, nor sufficient to initiate the EAE lesion. Nevertheless, these cells could provoke exacerbation of pathology if transferred into mice with ongoing EAE. The PAD2 and PAD4 enzymes were markedly upregulated in the inflamed CNS. Therefore, once inflammation is established, citrullination of target autoantigens can allow an expanded repertoire of T cells to contribute to CNS pathology.

Melatonin is an indoleamine widely distributed in the evolution that shows a great functional versatility, playing an important role as a transmitter of photoperiodic information and exhibiting antioxidant, oncostatic, anti-aging and immunomodulatory properties. In vertebrates, this molecule is produced by the pineal gland and other extrapineal sites. The present study was carried out to investigate the presence of melatonin in thymus and the possibility of an endogenous melatonin synthesis in this organ, in which T cells are matured. In this work, we demonstrate in humans and rats that thymus contains melatonin, expresses the mRNAs encoding N-acetyltransferase and hydroxyindol-O-methyltransferase, the two key enzymes of the melatonin synthesis, and has this biosynthetic machinery activated. In addition, rat thymocytes cultured for 24 h exhibited high levels of melatonin. The results presented here suggest that human and rat thymuses are able to synthesize melatonin, which could have intracrine, autocrine and paracrine functions.

Experimental autoimmune encephalomyelitis (EAE), the experimental model for multiple sclerosis (MS), is triggered by myelin-specific Th1 and Th17 cells. The immunomodulatory activities of melatonin have been shown to be beneficial under several conditions in which the immune system is exacerbated. Here, we sought to elucidate the basis of the melatonin protective effect on EAE by characterizing the T effector/regulatory responses, particularly those of the memory cell subsets. Melatonin was tested for its effect on Th1, Th17 and T regulatory (Treg) cells in the lymph nodes and CNS of immunodominant peptide of myelin oligodendrocyte glycoprotein (pMOG)-immunized and EAE mice, respectively. The capacity of melatonin to ameliorate EAE as well as modifying both T cell response and effector/regulatory balance was surveyed. T cell memory subsets and CD44, a key activation marker involved in the EAE pathogenesis, were also examined. Melatonin protected from EAE by decreasing peripheral and central Th1/Th17 responses and enhancing both the Treg frequency and IL-10 synthesis in the CNS. Melatonin reduced the T effector memory population and its pro-inflammatory response and regulated CD44 expression, which was decreased in T effector cells and increased in Tregs. The alterations in the T cell subpopulations were associated with a reduced mononuclear infiltration (CD4 and CD11b cells) of the melatonin-treated mice CNS. For the first time, we report that melatonin protects against EAE by controlling peripheral and central T effector/regulatory responses, effects that might be partially mediated by CD44. This immunomodulatory effect on EAE suggests that melatonin may represent an effective treatment option for MS.

To assess whether oxidative damage in some tissues was related to their melatonin concentration, endogenous melatonin levels and the age-linked protein and lipid damage in spleen, thymus and liver in 5-month-old SAM P8 mice were examined. The results show that high levels of melatonin in spleen and thymus correlate with lower protein and lipid damage. The liver, which had much lower melatonin concentrations than the other two tissues, had much higher levels of oxidatively damaged protein, as measured by carbonyl values. These results add new evidence concerning the protective role of endogenous melatonin as an antioxidant agent, and suggest that a treatment with this molecule might help to reduce age-associated functional deficits in many organs, including those of the immune system.

To gain insight into the relationship between thymus and pineal gland during rat development, the melatonin content as well as the activity and expression of the two key enzymes for melatonin biosynthesis, i.e. N-acetyltransferase (NAT) and hydroxyindole-O-methyltransferase (HIOMT), were studied in the thymus at fetal and postnatal stages. Moreover, melatonin-membrane receptor (MT1) expression was also analyzed. We found both the expression and activity of thymic NAT and HIOMT at 18 days of fetal life. Additionally, there is production of melatonin in the thymus as well as MT1 expression at this fetal age. These results show values higher in day-time than at night-time. The pineal gland begins to produce significant levels of melatonin around postnatal day 16, and this synthesis shows a circadian rhythm with high values during the dark period; therefore the nocturnal serum melatonin may inhibit thymic melatonin production. To document this, we report an increased melatonin content of the thymus in pinealectomized rats compared with sham-pinealectomized. In conclusion, these results show, for the first time, the presence of the biosynthetic machinery of melatonin and melatonin production in developing rat thymus and that the pineal gland may regulate this process.

Human lymphocytes have recently been described as an important physiological source of melatonin (N-acetyl-5-methoxytryptamine), which could be involved in the regulation of the human immune system. On the other hand, stimulation of IL-2 production by exogenous melatonin has been shown in the Jurkat human lymphocytic cell line. Furthermore, both melatonin membrane and nuclear receptors are present in these cells. In this study, we show that the necessary machinery to synthesize melatonin is present and active in resting and stimulated Jurkat cells. Accordingly, we have found that cells synthesize and release melatonin in both conditions. Therefore, we investigated whether endogenous melatonin produced by Jurkat cells was involved in the regulation of IL-2 production. When melatonin membrane and nuclear receptors were blocked using specific antagonists, luzindole and CGP 55644, respectively, we found that IL-2 production decreased, and this drop was reverted by exogenous melatonin. Additionally, PHA activation of Jurkat cells changed the profile of melatonin nuclear receptor mRNA expression. A previous study showed that exogenous melatonin is able to counteract the decrease in IL-2 production caused by prostaglandin E2 (PGE2) in human lymphocytes via its membrane receptor. In our model, when we blocked the melatonin membrane receptor with luzindole, the inhibitory effect of PGE2 on IL-2 production was higher. Therefore, we have demonstrated the physiological role of endogenous melatonin in this cell line. These findings indicate that endogenous melatonin synthesized by human T cells would contribute to regulation of its own IL-2 production, acting as an intracrine, autocrine, and/or paracrine substance.

Since melatonin was first isolated in 1958 up to the last few years, this substance was considered a hormone exclusive to the pineal gland. Although melatonin has lately been identified in a large number of extrapineal sites, its potential biological actions have not yet been studied. This paper shows that human lymphocyte-synthesized melatonin plays a crucial role modulating IL-2/IL-2 receptor system because when blocking melatonin biosynthesis by the tryptophan hydroxylase inhibitor, parachlorophenylalanine, both IL-2 and IL-2 receptor levels fell, restoring them by adding exogenous melatonin. Moreover, we demonstrated that this endogenous melatonin interfered with the exogenous melatonin effect on IL-2 production. Melatonin exerted these effects by a receptor-mediated action mechanism because both IL-2 and IL-2 receptor expressions significantly decreased when lymphocytes were incubated in the presence of the specific membrane and/or nuclear melatonin receptor antagonists, luzindole, and/or CGP 55644, respectively. Finally, we made the real significance of the membrane melatonin receptors in this process clear, so prostaglandin E2-induced inhibition on IL-2 production increased when we blocked the membrane receptors using luzindole. In conclusion, these data show that endogenous melatonin is an essential part for an accurate response of human lymphocytes through the modulation of IL-2/IL-2 receptor system.

This review summarizes the numerous observations published in recent years which have shown that one of the most significant of melatonin's pleiotropic effects is the regulation of the immune system. The overview summarizes the immune effects of pinealectomy and the association between rhythmic melatonin production and adjustments in the immune system as markers of melatonin's immunomodulatory actions. The effects of both in vivo and in vitromelatonin administration on non-specific, humoral, and cellular immune responses as well as on cellular proliferation and immune mediator production are presented. One of the main features that distinguishes melatonin from the classical hormones is its synthesis by a number of non-endocrine extrapineal organs, including the immune system. Herein, we summarize the presence of immune system-synthesized melatonin, its direct immunomodulatory effects on cytokine production, and its masking effects on exogenous melatonin action. The mechanisms of action of melatonin in the immune system are also discussed, focusing attention on the presence of membrane and nuclear receptors and the characterization of several physiological roles mediated by some receptor analogs in immune cells. The review focuses on melatonin's actions in several immune pathologies including infection, inflammation, and autoimmunity together with the relation between melatonin, immunity, and cancer.

Previous studies have shown that melatonin acts through specific receptors, including MT(1) and MT(2) membrane receptors as well as a nuclear receptor belonging to the orphan nuclear receptor family. Therefore, the goal of this study was to determine whether melatonin receptors mRNA is expressed in mouse peripheral tissues. To study the different receptors subtype expression, we have used a reverse-transcription polymerase chain reaction (RT-PCR) procedure followed by Southern hybridization with specific digoxigenin-labeled probes. RT-PCR studies revealed the presence of both MT(1) membrane receptors and ROR(alpha)1 nuclear receptors in all the peripheral tissues examined (brain, heart, lung, liver and kidney). Moreover, the expression of ROR(alpha)1 nuclear receptors was also demonstrated by Western-blot. In contrast, expression of MT(2) membrane receptors was only observed in brain and lung. These results suggest that melatonin, acting through its different subtypes receptors, plays a role in the neuroendocrine regulation of peripheral tissues function.

Melatonin could act on immune system by regulating cytokine production of immunocompetent cells. The hormone enhances IL-2, IFN-γ and IL-6 production by cultured human mononuclear cells. As enhancement of IL-6 production is related to monocyte activation by melatonin, the hormone acts on human lymphoid cells causing a Th1-type response. This paper shows that melatonin seems to promote a Th1-response by increasing IL-12 production. The hormone enhances IL-12 production by cultured monocytes under suboptimal stimulation in a dose-dependent way. The effect of the hormone increases when PBMCs are incubated with melatonin before monocyte isolation. Enhanced IL-12 production by melatonin can also be shown in cultured human mononuclear cells.

Nowadays, the effector capacity of melatonin in the immune system is indisputable. The almost ubiquitous distribution of melatonin receptors, its immune synthesis, and the effects of several immunological mediators on the melatonin production reinforce the concept of the melatonin/immune bidirectional circuit. Throughout the present chapter, we have highlighted that melatonin acts on the immune response by combined mechanisms which mainly involve the modulation of cytokines and the oxidative stress markers production. Overall, melatonin acts as an immune activator in basal or immunodepressed conditions, whereas it might exert a negative regulation under transient or chronic exacerbated immune response. Due to the variety of the melatonin immunomodulatory actions, it has been tested in extensive models. Thus, the clinical relevance of the several faces of melatonin on immune conditions such as infection, autoimmunity, vaccination, and immunosenescence as well as transplant and cancer is also reviewed.

The role of melatonin in glucose homeostasis is an active area of investigation. There is a growing body of evidence suggesting a link between disturbances in melatonin production and impaired insulin, glucose, lipid metabolism, and antioxidant capacity. Furthermore, melatonin has been found to influence insulin secretion both in vivo and in vitro, and night-time melatonin levels are related to night-time insulin concentrations in patients with diabetes. In several recent studies, a single nucleotide polymorphism of the human melatonin receptor 1B has been described as being causally linked to an increased risk of developing type 2 diabetes. Taken together, these data suggest that endogenous as well as exogenous melatonin may play a role in diabetes and associated metabolic disturbances not only by regulating insulin secretion but also by providing protection against reactive oxygen species, considering pancreatic β -cells are particularly susceptible to oxidative stress because they possess only low-antioxidative capacity.

It has been historically assumed that the pineal gland is the major source of melatonin (N-acetyl-5-methoxytryptamine) in vertebrates. Melatonin plays a central role in fine-tuning circadian rhythms in vertebrate physiology. In addition, melatonin shows a remarkable functional versatility exhibiting antioxidant, oncostatic, antiaging, and immunomodulatory properties. Melatonin has been identified in a wide range of organisms from bacteria to human beings. Its biosynthesis from tryptophan involves four well-defined intracellular steps catalyzed by tryptophan hydroxylase, aromatic amino acid decarboxylase, serotonin-N-acetyltransferase, and hydroxyindole-O-methyltransferase. Here, for the first time, we document that both resting and phytohemagglutinin-stimulated human lymphocytes synthesize and release large amounts of melatonin, with the melatonin concentration in the medium increasing up to five times the nocturnal physiological levels in human serum. Moreover, we show that the necessary machinery to synthesize melatonin is present in human lymphocytes. Furthermore, melatonin released to the culture medium is synthesized in the cells, because blocking the enzymes required for its biosynthesis or inhibiting protein synthesis in general produced a significant reduction in melatonin release. Moreover, this inhibition caused a decrease in IL-2 production, which was restored by adding exogenous melatonin. These findings indicate that in addition to pineal gland, human lymphoid cells are an important physiological source of melatonin and that this melatonin could be involved in the regulation of the human immune system, possibly by acting as an intracrine, autocrine, and/or paracrine substance.

CD4(+) T cells have a well-defined pathogenic role in experimental autoimmune encephalomyelitis, the rodent model of multiple sclerosis (MS), yet CD8(+) T cells are commonly found in MS lesions. To determine whether immunological tolerance might impact differently on CD4(+) versus CD8(+) T cells, we studied T cell responses in mice genetically deficient for the central nervous system (CNS) autoantigen myelin oligodendrocyte glycoprotein (MOG) versus wild type (WT) C57BL/6 mice. We show that MOG(-/-) mice have enhanced sensitivity to immunization with the immunodominant peptide of MOG (35-55), as evidenced by increased expansion of both CD4(+) and CD8(+) T cell subsets. Most strikingly, CD8(+) T cells from MOG(-/-) mice responded to a novel T cell epitope which binds to MHC class I with high affinity. Despite this, MOG-responsive CD8(+) T cells sourced from either WT or MOG(-/-) mice failed to initiate CNS inflammation upon transfer to MOG-sufficient mice. In our hands, this capacity was only found in CD4(+) T cells. However, MOG(-/-) CD4(+) cells did not show greater pathogenic activity than their WT counterparts. Our data indicate that, in the presence of endogenous MOG, CD8(+) T cells capable of responding to a MHC class I-restricted epitope that can be stably expressed are subject to rigorous control through central and/or peripheral tolerance.

Melatonin has been proposed as regulating the immune system by affecting cytokine production in immunocompetent cells, enhancing the production of several T helper (Th)1 cytokines. To further investigate the melatonin's role in IL-2/IL-2R system, we established an inducible T-REx expression system in Jurkat cells in which the protein levels of HIOMT enzyme or MT(1) receptor were significantly down-regulated upon tetracycline incubation. We found that T-REx Jurkat cells with lower levels of HIOMT activity, and consequently lower content of endogenous melatonin, showed IL-2 production decrease after activation with lectin. Likewise, tetracycline-inducible stable cell line expressing MT(1) antisense produced decreased amounts of IL-2 (mRNA and protein levels) after stimulation. Moreover, in T-Rex-MT(1) cells incubated with tetracycline, a sub-optimal PHA dose failed to induce the early activation marker CD25 on the cell surface. The results shown here support the relevance of endogenous melatonin and its signaling in T cell activation.

10 páginas, 7 figuras, 1 tabla., Human lymphocyte melatonin, through membrane and nuclear receptors binding, acts as an activator in IL-2 production. Antagonism of membrane melatonin receptors using luzindole exacerbates the drop of the IL-2 production induced by PGE2 in peripheral blood mononuclear and Jurkat cells. This paper studies the melatonin membrane and nuclear receptors interplay in PGE2-diminished IL-2 production. The decrease in IL-2 production after PGE2 and/or luzindole administration correlated with downregulation in the nuclear receptor RORα. We also highlighted a role of cAMP in the pathway, because forskolin mimicked the effects of luzindole and/or PGE2 in the RORα expression. Finally, a significant RORα downregulation was observed in T cells permanently transfected with inducible MT1 antisense. In conclusion, we show a novel connection between melatonin membrane receptor signalling and RORα expression, opening a new way to understand melatonin regulation in lymphocyte physiology., This work was supported by a grant from the Ministerio de Sanidad FIS (06/0091) and Red-FIS (RD06/0013/0001) and Grupo Excelencia del Plan Andaluz de Investigación (P06-CTS-01604).

Melatonin production is not restricted to the pineal gland. Several extrapineal sources of this indole such as retina, Harderian gland, and immune system are well documented. Melatonin of pineal origin is not present in the rat at early stages of development. To assess the potential capacity of local melatonin synthesis by the immature brain and to gain insight into the relationship between melatonin production by the brain (without the pineal gland) and pineal gland during rat development, the melatonin content as well as the expression and activity of the melatonin-synthesizing enzymes, N-acetyltransferase (NAT) and hydroxyindole-O-methyltransferase (HIOMT), were studied at fetal and postnatal stages. Moreover, melatonin-membrane receptor (MT(1)) expression was also analyzed. Both, the expression and activity of NAT and HIOMT were found in the brain with significant day/night differences in enzymes activities. Additionally, melatonin content was detected in all stages showing day/night differences depending on the stage of development. The brain nocturnal melatonin content was higher than diurnal content on postnatal day 16 and in adult rats which is in accordance with the pineal melatonin synthesis. To investigate the origin of this brain melatonin, pinealectomized rats were used and we found that the developing brain produced its own melatonin. Also, MT(1) expression was detected in brain during development. These results demonstrate that, when the pineal is not yet producing melatonin, there is melatonin synthesis by the brain that could be used as protection from free radical damage and/or could exert some actions through MT(1) receptors.

Septic shock, the most severe problem of sepsis, is a lethal condition caused by the interaction of a pathogen-induced long chain of sequential intracellular events in immune cells, epithelium, endothelium, and the neuroendocrine system. The lethal effects of septic shock are associated with the production and release of numerous pro-inflammatory biochemical mediators including cytokines, nitric oxide and toxic oxygen and nitrogen radicals, together with development of massive apoptosis. As melatonin has remarkable properties as a cytokine modulator, antioxidant and anti-apoptotic agent, the present study was designed to evaluate the possible protective effect of melatonin against LPS-induced septic shock in Swiss mice. We observed that intraperitoneally (i.p.) administered-melatonin (10 mg/kg) 30 min prior, and 1 hr after i.p. LPS injection (0.75 mg/animal) markedly protected mice from the LPS lethal effects with 90% survival rates for melatonin and 20% for LPS-injected mice after 72 hr. The melatonin effect was mediated by modulating the release of pro-/anti-inflammatory cytokine levels, protection from oxidative damage and counteracting apoptotic cell death. Melatonin was able to partially counteract the increase in LPS-induced pro-inflammatory cytokine levels such as tumor necrosis factor-alpha, IL-12 and interferon-gamma at the local site of injection, while it increased the production of the anti-inflammatory cytokine IL-10 both locally and systemically. Furthermore, melatonin inhibited the LPS-induced nitrite/nitrate and lipid peroxidation levels in brain and liver and counteracted the sepsis-associated apoptotic process in spleen. In conclusion, we have demonstrated that melatonin improves the survival of mice with septic shock via its pleiotropic functions as an immunomodulator, antioxidant and anti-apoptotic mediator.

The neurohormone melatonin plays a fundamental role in neuroimmunomodulation of several mammalian species, including mice. This effect is supported by the existence of specific melatonin-binding sites in murine immunocompetent organs. Moreover, using melatonin receptor analogues, several effects of the neurohormone on mice physiology through its membrane and nuclear receptors have been described. The expression of these receptors has never been studied, despite indirect evidence showing the presence of melatonin receptor in the murine immune system. At present, the MT1 and MT2 membrane receptors, and nuclear receptors belonging to the RZR/ROR family have been related to the immunomodulator effect of melatonin. Here, we show the presence of membrane and nuclear melatonin-binding sites in mouse thymus and spleen, using the specific melatonin membrane (S 20098) and nuclear (CGP 52608) receptor agonist. To confirm the presence of melatonin receptors, we analyzed the presence of membrane and nuclear receptor mRNA and protein by RT-PCR, Southern blot, and Western blot. Thus, we show that MT1 and RORalpha receptor mRNA and protein are expressed in both thymus and spleen, while MT2 receptor mRNA is only detected in the thymus. This expression of melatonin receptors strongly supports the idea of an immunomodulatory role of melatonin through its receptors.

In this chapter, we review the evidence accumulated during the last 10 years indicating the relationship between the pineal gland and its hormone melatonin and the immune system. A number of experiments have shown that melatonin has immunoenhancing properties that can be observed studying different immune functions in vivo as well as the production of cytokines and other immune messengers in vitro. Moreover, we report evidence for the presence of both typical membrane receptors (Mel1a) for melatonin as well as nuclear receptors in immunocompetent cells. Data presented suggest that the nuclear receptor is the primary mechanism of melatonin to participate in the regulation of cytokines by lymphocytes.

Melatonin, the chief secretory product of the pineal gland, is a potent and efficient endogenous radical scavenger. Thus, melatonin was shown to protect different biomolecules, such as DNA, membrane lipids, and cytosolic proteins, from oxidative damage induced by oxygen-derived free radicals. In order to study the protective role of melatonin in hydrogen peroxide (H 2 O 2 )-induced DNA damage. U-937 cells were treated with different concentrations of H 2 O 2 , either in the presence or absence of melatonin, and DNA damage was assessed using the cytokinesis-block micronucleus technique. Melatonin diminished H 2 O 2 -induced micronuclei production both in short and long treatments. Additionally, melatonin concentrations higher than I μM were capable of protecting cells from spontaneous micronuclei production. These data suggest that melatonin, an endogenous antioxidant and nontoxic compound, may have an important role in protecting cells from genetic damage due to free radicals, supporting the idea of this hormone as a possible therapeutic agent in preventing aging and age-related diseases.