ABSTRACT
Glucocorticoids and melatonin display immunomodulatory functions, with both immune-stimulatory and suppressor effects, depending on the context. While their immune properties are well-explored in mammals, there are still few studies on this immune-endocrine interaction in an inflammatory context in amphibians, all of them under captivity conditions, which can constitute a stressor for these animals. Evaluating how amphibians react to an immune challenge in the field would reveal relevant information regarding how immune-physiological parameters are modulated in natural conditions. This study aimed to investigate the effects of lipopolysaccharide (LPS) injection in male toads (Rhinella icterica) recently captured in their natural habitat in the Atlantic Forest at two different times of the day. We evaluated: splenic cytokines mRNA (interleukin [IL]-1ß, IL-6, IL-10, interferon-γ) and complement system protein (C1s), plasma bacterial killing ability (BKA), plasma corticosterone (CORT), melatonin (MEL), and testosterone (T) levels, and neutrophil to lymphocyte ratio (NLR), two hours post-injections. LPS-injection increased NLR, the gene expression of IL-1ß, and less evidently CORT levels independently of the time of the day. These results evidence LPS-induced inflammation, similarly observed in toads in captivity. Saline and LPS-injected toads showed a positive correlation between IL-1ß and IL-6, both cytokines with pro-inflammatory effects. Also, CORT was negatively associated with T, suggesting inhibition of the hypothalamus-pituitary-gonadal axis in the LPS-stimulated group. Our results are associated with the first stage of the inflammatory assemblage. Studies evaluating further steps of this process might lead to a better understanding of the immune-endocrine relations in amphibians.
Subject(s)
Lipopolysaccharides , Melatonin , Animals , Bufonidae/physiology , Corticosterone , Ecosystem , Interleukin-6 , Lipopolysaccharides/toxicity , Male , MammalsABSTRACT
Cultured cell lines are the workhorse of cancer research, but the extent to which they recapitulate the heterogeneity observed among malignant cells in tumors is unclear. Here we used multiplexed single-cell RNA-seq to profile 198 cancer cell lines from 22 cancer types. We identified 12 expression programs that are recurrently heterogeneous within multiple cancer cell lines. These programs are associated with diverse biological processes, including cell cycle, senescence, stress and interferon responses, epithelial-mesenchymal transition and protein metabolism. Most of these programs recapitulate those recently identified as heterogeneous within human tumors. We prioritized specific cell lines as models of cellular heterogeneity and used them to study subpopulations of senescence-related cells, demonstrating their dynamics, regulation and unique drug sensitivities, which were predictive of clinical response. Our work describes the landscape of heterogeneity within diverse cancer cell lines and identifies recurrent patterns of heterogeneity that are shared between tumors and specific cell lines.
Subject(s)
Cell Line, Tumor , Genetic Heterogeneity , Neoplasms/genetics , Precancerous Conditions/genetics , Cell Line, Tumor/drug effects , Cellular Senescence/genetics , Drug Screening Assays, Antitumor , Humans , RNA-Seq , Stress, Physiological/genetics , Tumor MicroenvironmentABSTRACT
Morning shift sleep restriction has been associated with higher plasma IL-6 levels. The aim of this study was to investigate the effect of sleep duration on salivary IL-6. Sleep duration of morning shift workers was estimated by actigraphy. Workers with "longer sleep duration" (LSD; N = 6) and "shorter sleep duration" (SSD; N = 15) were then compared regarding salivary IL-6 levels determined at 14:00 h, bed and wake times. SSD workers did not show daily variation of IL-6 and presented higher levels at bedtime and 14:00 h compared to LSD workers. In this study, SSD is associated with an increase in salivary IL-6 content.
Subject(s)
Circadian Rhythm/physiology , Interleukin-6/biosynthesis , Saliva/metabolism , Sleep/physiology , Wakefulness/physiology , Adult , Female , Humans , Hydrocortisone/metabolism , Male , Middle Aged , Time Factors , Work Schedule ToleranceABSTRACT
Adenosine triphosphate (ATP) is released onto the pinealocyte, along with noradrenaline, from sympathetic neurons and triggers P2Y1 receptors that enhance ß-adrenergic-induced N-acetylserotonin (NAS) synthesis. Nevertheless, the biotransformation of NAS into melatonin, which occurs due to the subsequent methylation by acetylserotonin O-methyltransferase (ASMT; EC 2.1.1.4), has not yet been evaluated in the presence of purinergic stimulation. We therefore evaluated the effects of purinergic signaling on melatonin synthesis induced by ß-adrenergic stimulation. ATP increased NAS levels, but, surprisingly, inhibited melatonin synthesis in an inverse, concentration-dependent manner. Our results demonstrate that enhanced NAS levels, which depend on phospholipase C (PLC) activity (but not the induction of gene transcription), are a post-translational effect. By contrast, melatonin reduction is related to an ASMT inhibition of expression at both the gene transcription and protein levels. These results were independent of nuclear factor-kappa B (NF-kB) translocation. Neither the P2Y1 receptor activation nor the PLC-mediated pathway was involved in the decrease in melatonin, indicating that ATP regulates pineal metabolism through different mechanisms. Taken together, our data demonstrate that purinergic signaling differentially modulates NAS and melatonin synthesis and point to a regulatory role for ATP as a cotransmitter in the control of ASMT, the rate-limiting enzyme in melatonin synthesis. The endogenous production of melatonin regulates defense responses; therefore, understanding the mechanisms involving ASMT regulation might provide novel insights into the development and progression of neurological disorders since melatonin presents anti-inflammatory, neuroprotective, and neurogenic effects.
Subject(s)
Adenosine Triphosphate/pharmacology , Melatonin/biosynthesis , Pineal Gland/metabolism , Acetylserotonin O-Methyltransferase/metabolism , Adenosine Triphosphate/metabolism , Animals , Female , Male , NF-kappa B/metabolism , Rats , Rats, Wistar , Receptors, Purinergic P2Y1/metabolism , Serotonin/analogs & derivatives , Serotonin/metabolism , Type C Phospholipases/metabolismABSTRACT
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.
Subject(s)
Cerebellum/metabolism , Encephalitis/metabolism , Melatonin/biosynthesis , Pineal Gland/metabolism , Animals , Cell Survival , Cerebellum/drug effects , Encephalitis/chemically induced , Infusions, Intraventricular , Lipopolysaccharides/administration & dosage , Male , Neuroglia/drug effects , Neuroglia/metabolism , Neurons/drug effects , Neurons/metabolism , Pineal Gland/drug effects , Rats , Rats, WistarABSTRACT
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.
ABSTRACT
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-alpha 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-alpha 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.
Subject(s)
Immunity, Innate/immunology , Neuroimmunomodulation/immunology , Neurosecretory Systems/immunology , Pineal Gland/metabolism , Animals , Humans , Interleukin-2/immunology , Interleukin-2/metabolism , Melatonin/immunology , Melatonin/metabolism , Paracrine Communication/immunology , Pineal Gland/immunology , T-Lymphocytes, Helper-Inducer/immunology , T-Lymphocytes, Helper-Inducer/metabolism , Tumor Necrosis Factor-alpha/immunology , Tumor Necrosis Factor-alpha/metabolismABSTRACT
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-alpha (TNF-alpha) 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-alpha. This inhibition was transient, and a preincubation of TNF-alpha 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.
Subject(s)
Circadian Rhythm/drug effects , Circadian Rhythm/physiology , Immunity/physiology , Melatonin/biosynthesis , Pineal Gland/drug effects , Pineal Gland/metabolism , Tumor Necrosis Factor-alpha/pharmacology , Animals , Feedback, Physiological/drug effects , Female , Male , Norepinephrine/pharmacology , Pineal Gland/immunology , Rats , Rats, Wistar , Tissue Culture TechniquesABSTRACT
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 microm) for 48 hr in the presence or absence of the glucocorticoid receptor (GR) antagonist, mifepristone (1.0 microm), the proteasome inhibitor, N-acetyl-leucinyl-leucinyl-norleucinal-H (ALLN, 12.5 microm) or the antagonist of the nuclear factor kappa B (NFkappaB), pyrrolidinedithiocarbamate (PDTC, 12.5 microm). 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 microm 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 NFkappaB nuclear translocation was demonstrated by electromobility shift assay. Therefore, corticosterone potentiates noradrenaline-induced melatonin and NAS production through GR inhibition of NFkappaB 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.