Pedro Augusto Carlos Magno Fernandes

The Immune-Pineal Axis: A Shuttle between Endocrine and Paracrine Melatonin Sources

The time course of the innate immunological response involves a pro-inflammatory phase followed by an anti-inflammatory phase. Pro-inflammatory responses serve as a defense against several stressor conditions, and sequential processes that shut down these responses are necessary to avoid exacerbation or the development of chronic diseases. In the present review, we put together recent data that show that the pineal gland is a player in bidirectional control of the inflammatory response. Healthy organisms stay in standby mode, ready to react. The nocturnal melatonin surge impairs the rolling and adherence of leukocytes to endothelial layers, limiting cell migration, and stimulates nocturnal production of IL-2 by T helper lymphocytes, exerting an immunostimulatory effect. Otherwise, the release of TNF-α from activated macrophages suppresses the nocturnal melatonin surge, allowing a full cell migration and inhibiting IL-2 production. In sequence, activated mononuclear and polymorphonuclear cells produce melatonin in a paracrine manner at the site of injury, which scavenges free radicals and collaborates to resolve the inflammatory response. The sequential diminution of TNF-α production is followed by the recovery of the nocturnal melatonin surge and IL-2 production. In summary, the immune-pineal axis, implicated in the sequential involvement of the melatonin produced by the pineal gland and immune-competent cells, is an integral participant of the innate immune response.

Effect of TNF-α on the melatonin synthetic pathway in the rat pineal gland: basis for a ‘feedback’ of the immune response on circadian timing

Abstract:  A retino‐hypothalamic‐sympathetic pathway drives the nocturnal surge of pineal melatonin production that determines the synchronization of pineal function with the environmental light/dark cycle. In many studies, melatonin has been implicated in the modulation of the inflammatory response. However, scant information on the feedback action of molecules present in the blood on the pineal gland during the time course of an inflammatory response is available. Here we analyzed the effect of tumor necrosis factor‐α (TNF‐α) and corticosterone on the transcription of the Aa‐nat, hiomt and 14‐3‐3 protein genes in denervated pineal glands of rats stimulated for 5 hr with norepinephrine, using real‐time reverse transcription‐polymerase chain reaction. The transcription of Aa‐nat, a gene encoding the key enzyme in melatonin biosynthesis, together with the synthesis of the melatonin precursor N‐acetylserotonin, was inhibited by TNF‐α. This inhibition was transient, and a preincubation of TNF‐α for more than 24 hr had no detectable effect. In fact, a protein(s) transcribed, later on, as shown by cycloheximide, was responsible for the reversal of the inhibition of Aa‐nat transcription. In addition, corticosterone induced a potentiation of norepinephrine‐induced Aa‐nat transcription even after 48 hr of incubation. These data support the hypothesis that the nocturnal surge in melatonin is impaired at the beginning of an inflammatory response and restored either during the shutdown of an acute response or in a chronic inflammatory pathology. Here, we introduce a new molecular pathway involved in the feedback of an inflammatory response on pineal activity, and provide a molecular basis for understanding the expression of circadian timing in injured organisms.

Corticosterone modulates noradrenaline-induced melatonin synthesis through inhibition of nuclear factor kappa B

Abstract:  In chronically inflamed animals, adrenal hormones exert a positive control on the secretion of melatonin by the pineal gland. In this paper, the mechanism of corticosterone as a modulator of melatonin and N‐acetylserotonin (NAS) was determined. Rat pineal glands in culture, stimulated for 5 hr with noradrenaline (10 nm), were previously incubated with corticosterone (1.0 nm–1.0 μm) for 48 hr in the presence or absence of the glucocorticoid receptor (GR) antagonist, mifepristone (1.0 μm), the proteasome inhibitor, N‐acetyl‐leucinyl‐leucinyl‐norleucinal‐H (ALLN, 12.5 μm) or the antagonist of the nuclear factor kappa B (NFκB), pyrrolidinedithiocarbamate (PDTC, 12.5 μm). Corticosterone potentiated noradrenaline‐induced melatonin and NAS production in a bell‐shaped manner. The increase in NAS (12.9 ± 2.7, n = 6 versus 34.3 ± 8.3 ng per pineal) and melatonin (16.3 ± 2.0, n = 6 versus 44.3 ± 12.9 ng per pineal) content induced by 1 μm corticosterone was blocked by mifepristone, and mimicked by ALLN and PDTC. The presence of GRs was shown by [3H]‐dexamethasone binding (0.30 ± 0.09 pmol/mg protein) and corticosterone inhibition of NFκB nuclear translocation was demonstrated by electromobility shift assay. Therefore, corticosterone potentiates noradrenaline‐induced melatonin and NAS production through GR inhibition of NFκB nuclear translocation. To the best of our knowledge, this is the first time that this relevant pathway for passive and acquired immune response is shown to modulate melatonin production in pineal gland.

TLR4 and CD14 receptors expressed in rat pineal gland trigger NFKB pathway

Abstract:  Nuclear factor‐kappa B (NFKB), a pivotal player in inflammatory responses, is constitutively expressed in the pineal gland. Corticosterone inhibits pineal NFKB leading to an enhancement of melatonin production, while tumor necrosis factor (TNF) leads to inhibition of Aa‐nat transcription and the production of N‐acetylserotonin in cultured glands. The reduction in nocturnal melatonin surge favors the mounting of the inflammatory response. Despite these data, there is no clear evidence of the ability of the pineal gland to recognize molecules that signal infection. This study investigated whether the rat pineal gland expresses receptors for lipopolysaccharide (LPS), the endotoxin from the membranes of Gram‐negative bacteria, and to establish the mechanism of action of LPS. Here, we show that pineal glands possess both CD14 and toll‐like receptor 4 (TLR4), membrane proteins that bind LPS and trigger the NFKB pathway. LPS induced the nuclear translocation of p50/p50 and p50/RELA dimers and the synthesis of TNF. The maximal expression of TNF in cultured glands coincides with an increase in the expression of TNF receptor 1 (TNFR1) in isolated pinealocytes. In addition, LPS inhibited the synthesis of N‐acetylserotonin and melatonin. Therefore, the pineal gland transduces Gram‐negative endotoxin stimulation by producing TNF and inhibiting melatonin synthesis. Here, we provide evidence to reinforce the idea of an immune‐pineal axis, showing that the pineal gland is a constitutive player in the innate immune response.

DAILY VARIATION OF CONSTITUTIVELY ACTIVATED NUCLEAR FACTOR KAPPA B (NFKB) IN RAT PINEAL GLAND

In mammals, the production of melatonin by the pineal gland is mainly controlled by the suprachiasmatic nuclei (SCN), the master clock of the circadian system. We have previously shown that agents involved in inflammatory responses, such as cytokines and corticosterone, modulate pineal melatonin synthesis. The nuclear transcription factor NFKB, detected by our group in the rat pineal gland, modulates this effect. Here, we evaluated a putative constitutive role for the pineal gland NFKB pathway. Male rats were kept under 12 h:12 h light-dark (LD) cycle or under constant darkness (DD) condition. Nuclear NFKB was quantified by electrophoretic mobility shift assay on pineal glands obtained from animals killed throughout the day at different times. Nuclear content of NFKB presented a daily rhythm only in LD-entrained animals. During the light phase, the amount of NFKB increased continuously, and a sharp drop occurred when lights were turned off. Animals maintained in a constant light environment until ZT 18 showed diurnal levels of nuclear NFKB at ZT15 and ZT18. Propranolol (20 mg/kg, i.p., ZT 11) treatment, which inhibits nocturnal sympathetic input, impaired nocturnal decrease of NFKB only at ZT18. A similar effect was observed in free-running animals, which secreted less nocturnal melatonin. Because melatonin reduces constitutive NFKB activation in cultured pineal glands, we propose that this indolamine regulates this transcription factor pathway in the rat pineal gland, but not at the LD transition. The controversial results regarding the inhibition of pineal function by constant light or blocking sympathetic neurotransmission are discussed according to the hypothesis that the prompt effect of lights-off is not mediated by noradrenaline, which otherwise contributes to maintaining low levels of nuclear NFKB at night. In summary, we report here a novel transcription factor in the pineal gland, which exhibits a constitutive rhythm dependent on environmental photic information. (Author correspondence: rpmarkus@usp.br)

Selective protection of the cerebellum against intracerebroventricular LPS is mediated by local melatonin synthesis.

Although melatonin is mainly produced by the pineal gland, an increasing number of extra-pineal sites of melatonin synthesis have been described. We previously demonstrated the existence of bidirectional communication between the pineal gland and the immune system that drives a switch in melatonin production from the pineal gland to peripheral organs during the mounting of an innate immune response. In the present study, we show that acute neuroinflammation induced by lipopolysaccharide (LPS) injected directly into the lateral ventricles of adult rats reduces the nocturnal peak of melatonin in the plasma and induces its synthesis in the cerebellum, though not in the cortex or hippocampus. This increase in cerebellar melatonin content requires the activation of nuclear factor kappa B (NF-κB), which positively regulates the expression of the key enzyme for melatonin synthesis, arylalkylamine N-acetyltransferase (AA-NAT). Interestingly, LPS treatment led to neuronal death in the hippocampus and cortex, but not in the cerebellum. This privileged protection of cerebellar cells was abrogated when G-protein-coupled melatonin receptors were blocked by the melatonin antagonist luzindole, suggesting that the local production of melatonin protects cerebellar neurons from LPS toxicity. This is the first demonstration of a switch between pineal and extra-pineal melatonin production in the central nervous system following a neuroinflammatory response. These results have direct implications concerning the differential susceptibility of specific brain areas to neuronal death.

Long-Lasting Priming of Endothelial Cells by Plasma Melatonin Levels

Background Endothelial cells are of great interest for cell therapy and tissue engineering. Understanding the heterogeneity among cell lines originating from different sources and culture protocols may allow more standardized material to be obtained. In a recent paper, we showed that adrenalectomy interferes with the expression of membrane adhesion molecules on endothelial cells maintained in culture for 16 to 18 days. In addition, the pineal hormone, melatonin, reduces the adhesion of neutrophils to post-capillary veins in rats. Here, we evaluated whether the reactivity of cultured endothelial cells maintained for more than two weeks in culture is inversely correlated to plasma melatonin concentration. Methodology/Principal Findings The nocturnal levels of melatonin were manipulated by treating rats with LPS. Nocturnal plasma melatonin, significantly reduced two hours after LPS treatment, returned to control levels after six hours. Endothelial cells obtained from animals that had lower nocturnal melatonin levels significantly express enhanced adhesion molecules and iNOS, and have more leukocytes adhered than cells from animals that had normal nocturnal levels of melatonin (naïve or injected with vehicle). Endothelial cells from animals sacrificed two hours after a simultaneous injection of LPS and melatonin present similar phenotype and function than those obtained from control animals. Analyzing together all the data, taking into account the plasma melatonin concentration versus the expression of adhesion molecules or iNOS we detected a significant inverse correlation. Conclusions/Significance Our data strongly suggest that the plasma melatonin level primes endothelial cells “in vivo,” indicating that the state of the donor animal is translated to cells in culture and therefore, should be considered for establishing cell banks in ideal conditions.

Molecular Basis for Defining the Pineal Gland and Pinealocytes as Targets for Tumor Necrosis Factor

The pineal gland, the gland that translates darkness into an endocrine signal by releasing melatonin at night, is now considered a key player in the mounting of an innate immune response. Tumor necrosis factor (TNF), the first pro-inflammatory cytokine to be released by an inflammatory response, suppresses the translation of the key enzyme of melatonin synthesis (arylalkylamine-N-acetyltransferase, Aanat). Here, we show that TNF receptors of the subtype 1 (TNF-R1) are expressed by astrocytes, microglia, and pinealocytes. We also show that the TNF signaling reduces the level of inhibitory nuclear factor kappa B protein subtype A (NFKBIA), leading to the nuclear translocation of two NFKB dimers, p50/p50, and p50/RelA. The lack of a transactivating domain in the p50/p50 dimer suggests that this dimer is responsible for the repression of Aanat transcription. Meanwhile, p50/RelA promotes the expression of inducible nitric oxide synthase (iNOS) and the production of nitric oxide, which inhibits adrenergically induced melatonin production. Together, these data provide a mechanistic basis for considering pinealocytes a target of TNF and reinforce the idea that the suppression of pineal melatonin is one of the mechanisms involved in mounting an innate immune response.

Amyloid β peptide directly impairs pineal gland melatonin synthesis and melatonin receptor signaling through the ERK pathway

Melatonin is the hormone produced by the pineal gland known to regulate physiologic rhythms and to display immunomodulatory and neuroprotective properties. It has been reported that Alzheimer disease patients show impaired melatonin production and altered expression of the 2 G protein‐coupled melatonin receptors (MTRs), MT1 and MT2, but the underlying mechanisms are not known. Here we evaluated whether this dysfunction of the melatonergic system is directly caused by amyloid β peptides (Aβ1‐40 and Aβ1‐42). Aβ treatment of rat pineal glands elicited an inflammatory response within the gland, evidenced by the up‐regulation of 52 inflammatory genes, and decreased the production of melatonin up to 75% compared to vehicle‐treated glands. Blocking NF‐κB activity prevented this effect. Exposure of HEK293 cells stably expressing recombinant MT1 or MT2 receptors to Aβ lead to a 40% reduction in [125I]iodomelatonin binding to MT1. ERK1/2 activation triggered by MTRs, but not by the β2‐adrenergic receptor, was markedly impaired by Aβ in HEK293 transfected cells, as well as in primary rat endothelial cells expressing endogenous MTRs. Our data reveal the melatonergic system as a new target of Aβ, opening new perspectives to Alzheimer disease diagnosis and therapeutic intervention.—Cecon, E., Chen, M., Marçola, M., Fernandes, P. A. C., Jockers, R., Markus, R. P. Amyloid β peptide directly impairs pineal gland melatonin synthesis and melatonin receptor signaling through the ERK pathway. FASEB J. 29, 2566‐2582 (2015). www.fasebj.org

Melatoninergic System in Parkinson’s Disease: From Neuroprotection to the Management of Motor and Nonmotor Symptoms

Melatonin is synthesized by several tissues besides the pineal gland, and beyond its regulatory effects in light-dark cycle, melatonin is a hormone with neuroprotective, anti-inflammatory, and antioxidant properties. Melatonin acts as a free-radical scavenger, reducing reactive species and improving mitochondrial homeostasis. Melatonin also regulates the expression of neurotrophins that are involved in the survival of dopaminergic neurons and reduces α-synuclein aggregation, thus protecting the dopaminergic system against damage. The unbalance of pineal melatonin synthesis can predispose the organism to inflammatory and neurodegenerative diseases such as Parkinsons disease (PD). The aim of this review is to summarize the knowledge about the potential role of the melatoninergic system in the pathogenesis and treatment of PD. The literature reviewed here indicates that PD is associated with impaired brain expression of melatonin and its receptors MT1 and MT2. Exogenous melatonin treatment presented an outstanding neuroprotective effect in animal models of PD induced by different toxins, such as 6-hydroxydopamine (6-OHDA), 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine (MPTP), rotenone, paraquat, and maneb. Despite the neuroprotective effects and the improvement of motor impairments, melatonin also presents the potential to improve nonmotor symptoms commonly experienced by PD patients such as sleep and anxiety disorders, depression, and memory dysfunction.