Lipopolysaccharide (LPS): potent activator and regulatior of gene transcription through NFKB signaling

LPS is the main component from membrane surface of gram-negative bacteria known as a potent activator of immune response. This effect is mediated mainly through the NFKB signaling pathway. NFKB remains inactivated in cytoplasm due to interactions with repressor proteins and the triggering of LPS receptors allows NFKB dimmers to translocate to the nucleus and to promote gene transcription. Besides the response activated by LPS being well understood, the regulatory steps of this response remain obscure. Is this review, we will describe how LPS activates NFKB pathway and the main steps that regulate this signaling with focus on the regulation of gene expression.O LPS é o principal componente da membrana externa de bactérias gram-negativas e conhecido como importante ativador da resposta imunológica. Seus efeitos são mediados principalmente pelo NFKB, um fator de transcrição que se mantém inativo no citoplasma e que migra para o núcleo após a interação do LPS com seus receptores, promovendo a transcrição de diversos genes relacionados à resposta inflamatória aguda. Embora a resposta frente ao LPS seja bem compreendida, pouco se sabe a respeito dos pontos de regulação dessa resposta. Nesse sentido, descreveremos como o LPS se comunica com o meio intracelular, revisaremos os principais pontos conhecidos sobre a regulação da expressão de genes induzidos pelo NFKB e a participação desse fator e dos receptores de LPS na resposta inflamatóri

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

Glia-Pinealocyte Network: The Paracrine Modulation of Melatonin Synthesis by Tumor Necrosis Factor (TNF)

The pineal gland, a circumventricular organ, plays an integrative role in defense responses. The injury-induced suppression of the pineal gland hormone, melatonin, which is triggered by darkness, allows the mounting of innate immune responses. We have previously shown that cultured pineal glands, which express toll-like receptor 4 (TLR4) and tumor necrosis factor receptor 1 (TNFR1), produce TNF when challenged with lipopolysaccharide (LPS). Here our aim was to evaluate which cells present in the pineal gland, astrocytes, microglia or pinealocytes produced TNF, in order to understand the interaction between pineal activity, melatonin production and immune function. Cultured pineal glands or pinealocytes were stimulated with LPS. TNF content was measured using an enzyme-linked immunosorbent assay. TLR4 and TNFR1 expression were analyzed by confocal microscopy. Microglial morphology was analyzed by immunohistochemistry. In the present study, we show that although the main cell types of the pineal gland (pinealocytes, astrocytes and microglia) express TLR4, the production of TNF induced by LPS is mediated by microglia. This effect is due to activation of the nuclear factor kappa B (NF-kB) pathway. In addition, we observed that LPS activates microglia and modulates the expression of TNFR1 in pinealocytes. As TNF has been shown to amplify and prolong inflammatory responses, its production by pineal microglia suggests a glia-pinealocyte network that regulates melatonin output. The current study demonstrates the molecular and cellular basis for understanding how melatonin synthesis is regulated during an innate immune response, thus our results reinforce the role of the pineal gland as sensor of immune status

Characterization of immune-pineal axis: pineal gland as a sensor for lipopolysaccharide (LPS)

O fator de transcrição nuclear kappa B (NFKB), central na resposta inflamatória, é constitutivamente expresso em glândulas pineais de rato. A inibição da translocação nuclear deste fator em pineais de rato por corticosterona potencia, enquanto que a inibição pela citocina fator de necrose tumoral (TNF) inibe a síntese de melatonina por inibição da transcrição da Aa-nat. Esta redução da produção noturna de melatonina está implicada em favorecer a montagem da resposta inflamatória. Embora dados da literatura sugerirem redução da produção de melatonina durante processos infecciosos, não há evidências diretas da habilidade da glândula pineal em reconhecer o lipopolissacarídeo (LPS), a endotoxina da membrana de bactérias gram-negativas. Esta dissertação investigou se a glândula pineal de ratos expressa receptores para o reconhecimento do LPS e estabeleceu possíveis mecanismos de ação desta endotoxina na glândula pineal de ratos. Nossos resultados demonstram que a glândula pineal expressa de maneira constitutiva os receptores CD14 e o TLR4. LPS induz a translocação nuclear dos dímeros p50/p50 e p50/RelA e a síntese de TNF em glândulas cultivadas. A máxima produção de TNF no meio de cultura é coincidente com a máxima expressão do receptor TNFR1 em pinealócitos. Além disso, LPS inibe a síntese de N-acetilserotonina e melatonina. Em conclusão, neste estudo, demonstramos que a pineal é alvo para o componente de bactérias gram-negativas LPS, reforçando a proposta de que esta glândula reconhece e gera respostas a moléculas que sinalizam a montagem da resposta inflamatória.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 (NAS) in cultured glands. The reduction of 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 possesses 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

Characteristics of toll-like receptors in rat pineal glands the and involvement in the understanding of the immune-pineal axis

A glândula pineal regula diversos ritmos biológicos e respostas de defesa em indivíduos hígidos através da síntese noturna de melatonina. Por outro lado, é sabido que processos inflamatórios reduzem a produção deste hormônio na glândula pineal. Neste estudo utilizamos um arranjo de qPCR para investigar a expressão circadiana de 84 genes relacionados a sinalização via receptores do tipo toll e NF-κB em glândulas pineais de rato. Confirmamos ainda, a expressão de 14 proteínas em pinealócitos através de ensaios de imunocitoquímica. Nossos resultados indicam que 70 genes inflamatórios são expressos ritmicamente na glândula pineal de ratos, enquanto 7 não são expressos e outros 7 genes são expressos, mas sem ritmicidade. Grande parte dos genes examinados apresenta padrão de expressão circadiana com maior conteúdo transcricional na fase de claro, atingindo a máxima expressão no final desta fase. Após o apagar das luzes, a expressão destes genes é rapidamente reduzida. Um possível envolvimento do ritmo de glicocorticóides endógeno sobre o padrão dessa expressão gênica foi avaliado através do bloqueio de GR por mifepristona, o que induziu a regulação da expressão de 13 genes e redução do conteúdo plasmático de melatonina no ZT18. Ainda, avaliamos o efeito da ativação dos TLR1, TLR2 e TLR6 sobre a glândula pineal e observamos que zimosan e Pam3CSK4 ativam a via do fator de transcrição NF-κB e bloqueia a síntese de melatonina induzida por noradrenalina in vitro. Por fim, utilizamos o modelo de obseidade induzido por dieta hipercalórica para avaliar se o processo inflamatório de baixa intensidade regula a síntede se melatonina in vivo. Observamos que nestas condições, a dieta hipercalórica induz rápido aumento no peso corporal e redução da produção noturna de melatonina. O efeito protetor da melatonina sobre o ganho de peso induzido por dieta foi testado através da restauração da melatonina na água de beber noturna de animais expostos à dieta. Em conjunto, nossos resultados indicam que genes inflamatórios são expressos ritmicamente na glândula pineal de ratos e influenciam a produção circadiana de melatonina via reconhecimento de padrões moleculares associados à patógenos ou sinais de perigoThe pineal gland regulates several circadian rhythms as well as immune responses in healthy animals via rhythmic production of melatonin, the hormone of darkness. On the other hand, nocturnal melatonin levels are reduced in the course of inflammation. To date, it remains to be clear the mechanisms by which the immune system affects pineal melatonin synthesis. Here we used a qPCR array profiler to investigate circadian gene expression of 84 genes related to Toll-Like Receptors and Nuclear Factor kappa B signaling. We also examined the expression of 14 proteins in pinealocytes by immunocytochemistry. Our results indicate rhythmic expression of 70 inflammatory genes, while 7 genes were not expressed and 7 were expressed without rhythmicity. The overall majority of genes tested showed a pattern of expression with a cumulative diurnal increase that peaks at the light phase of ZT12 followed by a fast reduction in the expression as soon as the light is turned off. The possible involvement of endogenous glucocorticoid rhythm in the modulation of pineal\'s inflammatory gene expression were tested by blocking Glucocorticoid Receptor (GR) using mifepristone. This procedure modulated the expression of 13 genes. In addition, the blockade of GR reduced the circulating melatonin levels at ZT18. The activation of TLR1, TLR2 and TLR6 induces the nuclear translocation of NF-κB signaling and blocks noradrenaline-induced melatonin synthesis in vitro. In addition, high-fat diet feeding increases body weight and reduce the circulating melatonin levels at ZT18. The protective role of melatonin in diet-induced weight gain was also determined by giving these rats melatonin in their drinking water at night. Altogether, our results highlight that inflammatory genes are transiently expressed in the rat pineal gland and influences the daily fluctuation of melatonin synthesi

Os ritmos circadianos e a reprodução em mamíferos

Apresenta-se uma revisão sobre o estudo da Cronobiologia com ênfase nos ritmos circadianos e a relação desses com fenômenos reprodutivos nos mamíferos

Procedimentos dolorosos e manejo da dor em recém-nascidos hospitalizados em unidade de terapia intensiva*

Objective: To characterize painful procedures, analgesic strategies, vital signs, and pain scores in hospitalized newborns. Method: This is a primary, observational, prospective clinical study, developed in a Brazilian public hospital. Demographic data, painful procedures, pain relief measures, vital signs, and pain scores were collected from the clinical records of 90 newborns admitted to the intensive care unit and evaluated between admission and the third day of admission. For statistical analysis, the software Statistic Package for the Social Sciences and the R Software were used. Results: Newborns underwent 2,732 painful procedures, 540 non-pharmacological and 216 pharmacological strategies. The most frequently performed procedure was the heel prick (20.96%). The most commonly recorded non-pharmacological strategy was dim lighting (28.33%) and continuous fentanyl (48.83%) was the main pharmacological measure adopted. Pain score and vital signs show variability in the period evaluated. Conclusion: Despite the high number of painful procedures, pain assessment records do not reflect procedural pain and the use of analgesic strategies was insufficient.Objetivo: Caracterizar los procedimientos dolorosos, estrategias analgésicas, señales vitales y los scores de dolor en recién nascidos hospitalizados. Método: Estudio clínico primario, de observación, prospectivo, desarrollado en un hospital público brasileño. Datos demográficos, procedimientos dolorosos, medidas de alivio del dolor, señales vitales y scores de dolor fueron seleccionados de las historias clínicas de 90 recién nacidos admitidos en la unidad de cuidados intensivos y evaluados entre la admisión y el tercer día de hospitalización. Para el análisis estadístico fueron utilizados el programa Statistic Package for the Social Sciences y el Software R. Resultados: Los recién nacidos fueron sometidos a 2.732 procedimientos dolorosos, 540 estrategias no farmacológicas y 216 farmacológicas. El procedimiento más realizado fue la punción del talón (20,96%). La estrategia no farmacológica más común fue la reducción de luminosidad (28,33%) y el fetanyl continuo (48,83%) fue la principal medida farmacológica adoptada. Conclusión: A pesar del número elevado de procedimientos dolorosos, los registros de evaluación del dolor no reflejan el dolor procedural y el uso de las estrategias analgésicas fue insuficiente.Objetivo: Caracterizar os procedimentos dolorosos, estratégias analgésicas, sinais vitais e os escores de dor em recém-nascidos hospitalizados. Método: Estudo clínico primário, observacional, prospectivo, desenvolvido em um hospital público brasileiro. Dados demográficos, procedimentos dolorosos, medidas de alívio da dor, sinais vitais e escores de dor foram coletados dos prontuários clínicos de 90 recém-nascidos admitidos na unidade de terapia intensiva e avaliados entre a admissão e o terceiro dia de internação. Para a análise estatística foram utilizados o programa Statistic Package for the Social Sciences e o Software R. Resultados: Os recém-nascidos foram submetidos a 2.732 procedimentos dolorosos, 540 estratégias não farmacológicas e 216 farmacológicas. O procedimento mais realizado foi a lancetagem de calcâneo (20,96%). A estratégia não farmacológica mais comumente registrada foi a redução de luminosidade (28,33%) e o fentanil contínuo (48,83%) foi a principal medida farmacológica adotada. O escore de dor e os sinais vitais apresentam variabilidade no período avaliado. Conclusão: A despeito do número elevado de procedimentos dolorosos, os registros de avaliação da dor não refletem a dor procedural e o uso das estratégias analgésicas foi insuficiente

Possible Role of Pineal and Extra-Pineal Melatonin in Surveillance, Immunity, and First-Line Defense

Melatonin is a highly conserved molecule found in prokaryotes and eukaryotes that acts as the darkness hormone, translating environmental lighting to the whole body, and as a moderator of innate and acquired defense, migration, and cell proliferation processes. This review evaluates the importance of pineal activity in monitoring PAMPs and DAMPs and in mounting an inflammatory response or innate immune response. Activation of the immune–pineal axis, which coordinates the pro-and anti-inflammatory phases of an innate immune response, is described. PAMPs and DAMPs promote the immediate suppression of melatonin production by the pineal gland, which allows leukocyte migration. Monocyte-derived macrophages, important phagocytes of microbes, and cellular debris produce melatonin locally and thereby initiate the anti-inflammatory phase of the acute inflammatory response. The role of locally produced melatonin in organs that directly contact the external environment, such as the skin and the gastrointestinal and respiratory tracts, is also discussed. In this context, as resident macrophages are self-renewing cells, we explore evidence indicating that, besides avoiding overreaction of the immune system, extra-pineal melatonin has a fundamental role in the homeostasis of organs and tissues

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 (naive 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 fromcontrol 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.FAPESP[07/07871-6]CNPq[472881/2009-4

Melatonin regulates the lipopolysaccharide-induced iNOS expression in endothelial cells.

<p>iNOS expression in cultures obtained from naïve (black bar), vehicle injected (white bars), lipopolysaccharide injected (LPS, 0.5 mg/kg; grey bars) or LPS + melatonin injected (MEL 3.0 mg/kg; grey diagonal striped bars) animals was determined by confocal immunofluorescence. The injections were performed 2 or 6 hours before sacrifice (6 hours after lights off –24h00). Data are expressed as mean ± SEM, n = 4–8 per group; *significantly different (P<0.05) versus 2 hours vehicle group (white bars).</p