Thesis (Ph. D.)--Ohio State University, 2007. / Title from first page of PDF file. Includes bibliographical references (p. 114-137).

Submitted by Hiolanda Rêgo (hiolandar@gmail.com) on 2013-08-14T18:53:05Z No. of bitstreams: 1 Tese_ICS_Bruno Penas Seara Pitanga.pdf: 3443977 bytes, checksum: 4b90d8bbc0ba9b0c3ac164075485f76e (MD5) / Made available in DSpace on 2013-08-14T18:53:05Z (GMT). No. of bitstreams: 1 Tese_ICS_Bruno Penas Seara Pitanga.pdf: 3443977 bytes, checksum: 4b90d8bbc0ba9b0c3ac164075485f76e (MD5) / CAPES;CNPq / A capacidade reduzida da resposta imunológica no microambiente do glioblastoma é principalmente devido à interação microglia/glioma e está relacionada à sua agressividade. Nossos estudos anteriores demonstraram que os flavonoides derivados de plantas podem atuar como inibidores de crescimento de linhagens celulares humanas de glioblastoma, em especial o flavonoide rutina inibe o crescimento e apresenta capacidade morfogênica, ainda estimula a libertação de agentes imunomoduladores como TNF e NO por astrócitos e microglia em culturas primárias de ratos. O objetivo desse estudo foi verificar a viabilidade de linhagens celulares de glioblastoma humano tratadas com flavonoides, bem como caracterizar essas células quanto à expressão de proteínas do citoesqueleto e morfologia, ainda, investigar se os flavonoides rutina e quercetina, nas concentrações de 50μM e 100μM, podem modular a resposta imunológica em co-cultura microglia/glioma. Como demonstrado através do teste MTT, após 72h de exposição com os flavonoides, ocorreu inibição dose-dependente da viabilidade das células TG1, U251 e GL15. A caracterização dessas linhagens revelou marcação conjunta de GFAP, beta III tubulina e Nestina em diferentes intensidades e localização. Além disso, verificamos diminuição da capacidade de formação de esferóides em culturas de células TG1 tratadas com rutina. Em culturas de célula C6 a viabilidade não foi afetada após a exposição direta aos flavonoides rutina e quercetina nas concentrações de 50 - 100μM por 48h. O teste azul de tripan revelou uma redução dessa viabilidade em ambos os flavonoides testados diretamente. Já durante exposição indireta de meios provenientes de culturas de microglia tratadas com rutina, observamos redução na viabilidade em C6. Além disso, observamos um aumento na expressão de OX-42 em culturas de microglia após a exposição com flavonoides rutina e quercetina, indicando sua ativação. Foi realizado em co-culturas microglia/glioma a marcação para isolectina B4, verificamos que o flavonoide rutina induziu aumento na proliferação de microglia após contagem das células isolectina positivas. Ainda, o teste ELISA realizada em culturas microgliais mostrou que os níveis de TNF foram aumentados em culturas tratadas com rutina 100μM ou quercetina 50- 100μM. A dosagem de nitrido mostrou aumento significativo somente após tratamento com quercetina 100μM. Através da citometria verificamos redução de pSTAT3 e NFkB em culturas de C6 e a análise ultraestrutural revelou figura de mielina, vacúolo fagocítico durante interação microglia/glioma. Verificamos ainda que houve aumento na intensidade da marcação de células apoptóticas quando tratadas com quercetina 100μM em gliomas, já com rutina ocorreu sutil marcação de anexina também em gliomas. Para quantificação dos vacúolos autofágicos em gliomas, utilizamos o corante fluorescente laranja de acridina. Em rutina 100 μM observamos aumento na intensidade de fluorescência, já o tratamento com quercetina verificamos aumento discreto na formação dos vacúolos autofágicos. Diversos estudos demonstram que o perfil de resposta Th1 está associado com um melhor prognóstico em pacientes com câncer. Esses achados sugerem que quercetina e rutina podem induzir mudanças no perfil regulatório da resposta microglial durante a interação com glioma, no entanto o flavonoide rutina mostrou um potencial melhor como imunomodulador. O entendimento da interação microglia/glioma será útil na elaboração de novas terapias baseadas na resposta imune contra o glioblastoma. / Salvador

京都大学 / 0048 / 新制・課程博士 / 博士(医学) / 甲第20237号 / 医博第4196号 / 新制||医||1019(附属図書館) / 京都大学大学院医学研究科医学専攻 / (主査)教授 松原 和夫, 教授 渡邉 大, 教授 伊佐 正 / 学位規則第4条第1項該当 / Doctor of Medical Science / Kyoto University / DGAM

No description available.

Ethanol (EtOH) abuse is the third leading cause of preventable death in the United States. Mounting evidence indicates that EtOH-induced neuropathology may result from multicellular responses in which microglia cells play a prominent role. Purinergic receptor signaling plays a key role in regulating microglial function and, more importantly, mediates EtOH-induced effects. In our current study we sought to determine the effects of EtOH on microglial cell function, specifically the role of purinergic receptor X4 (P2X4) in EtOH-mediated microglial responses. Our results show a significant up-regulation of P2X4 gene expression as analyzed by real-time qPCR and protein expression as analyzed by flow cytometry in embryonic stem cell-derived microglial cells (ESdM) after 48 hours of EtOH treatment, as compared to untreated controls. Calcium mobilization in EtOH treated ESdM cells was found to be P2X4R- dependent using 5-BDBD, a selective P2X4R antagonist. Blocking P2X4R signaling with 5-BDBD decreased the level of calcium mobilization compared to EtOH treatment alone. EtOH decreased migration of microglia towards fractalkine (CX3CL1) by 75% following 48 hours of treatment compared to control. CX3CL1-dependent migration was confirmed to be P2X4 receptor-dependent using the antagonist 5-BDBD, which reversed the effects as compared to EtOH alone. Similarly, 48 hours of EtOH treatment significantly decreased phagocytosis of microglia by 15% compared to control. 5-BDBD pre-treatment prior to EtOH treatment significantly increased microglial phagocytosis. These findings demonstrate that P2X4 receptor may play a role in modulating important regulatory functions in microglia in the context of EtOH abuse. P2X4R plays an important regulatory function in microglia. P2X4 is involved in a myriad of molecular signaling such as proliferation, activation of transcription factors, specifically through the MAPK pathway, and ATP signaling. Here, we also investigated the intracellular signal transduction pathway that influences P2X4R expression in microglia in response to EtOH. We found EtOH (100 mM) decreased phosphorylated AKT and extracellular signal-regulated kinase (ERK) cascades in ESdM cells. EtOH effect on ERK phosphorylation was completely inhibited by U0126, an inhibitor of MEK 1 and 2. However, PD98095, a potent inhibitor of MEK1 but a weak inhibitor of MEK2 had modest effect on phosphorylated ERK1/2 suggesting a possible role of MEK2-dependent ERK signaling in modulating EtOH induced effects on microglia. Utilization of 5-BDBD, a selective P2X4R antagonist reversed the EtOH-induced effect on phosphorylated AKT and ERK. Next we wanted to examine the effects of EtOH on transcription factor activity to determine the signaling mediators, which may play a role in EtOH-induced increase in P2X4R in microglia. EtOH increased transcriptional activity of NFκB, NFAT, and CREB,, however 5-BDBD blocked the effect on CREB transcriptional activity alone, suggesting a specific role of CREB in EtOH-induced expression of P2X4R in microglia. In summary, EtOH affects the expression of P2X4R in microglial cells and contributes to aberrant microglial effector function including phagocytosis and migration as well as alterations in calcium mobilization. Furthermore, pharmacological blockade with a selective P2X4R antagonist reversed the action, suggesting that P2X4R may play a role in mediating EtOH-induced effects on microglia. EtOH decreased expression of ERK and AKT, which was blocked with the P2X4R antagonist, suggesting EtOH effect may contribute to irregular microglial signaling. Investigations regarding transcription factor NFκB, NFAT and CREB activity in response to EtOH, all showed an increase after EtOH treatment, however P2X4R antagonist only had an effect on CREB, blocking the effect of EtOH on its activity. Determining the mechanism underlying EtOH-induced increase in P2X4R expression still remains unclear. This research was conducted to investigate the importance of P2X4R signaling in EtOH-mediate microglial function. Although many more questions remain unanswered, these experiments have provided evidence to target purinergic receptor X4 as a potential mediator of EtOH-induced effects in microglia. / Pathology

Major Depressive Disorder is a common mood disorder, affecting more than 300 million people worldwide. Stress, being a potent trigger for various mood disorders, including depression, affects both the peripheral and central immune systems. Microglia are the resident immune cells in the brain and changes in their morphology and state of activation in response to stress have been reported in a number of brain regions, however most studies examined only male subjects. In our studies we investigated morphological changes in microglia in the nucleus accumbens and subregions of the hippocampus in both male and female mice following variable stress. Our findings demonstrate that following 6 days of variable stress female microglia acquire a more activated phenotype compared to microglia in males. These sex specific effects in microglia activation were also observed in the nucleus accumbes, but not the dentate gyrus after 28 days of stress. Utilizing a number of pharmacological agents, we further investigated whether changes in the inflammatory states of microglia modulate behavior in female mice in a variable stress model for depression. We found that rosiglitazone, peroxisome proliferator activated receptor gamma PPAR) agonist, which causes microglial shift to an anti-inflammatory state, conferred social resilience in female mice after 6 days of variable stress. We further demonstrated that blocking pro-inflammatory activation of microglia with minocycline, a tetracyclic antibiotic, did not prevent the stress effects on behavior. Surprisingly, prior pro-inflammatory activation of microglia with lipopolysaccharide (LPS), an endotoxin from gram-negative bacteria, was protective against subsequent variable stress. Finally, I describe the impact of systemic application of these drugs on microglia morphology and activation state, and cytokine levels in the nucleus accumbens of female mice. Taken together this body of work contributes to a growing number of studies demonstrating neuro-immune mechanisms associated with depression. / Doctor of Philosophy / Major Depressive Disorder is a debilitating mood disorder, affecting more than 300 million people worldwide. The prevalence of MDD is higher in women than men, and women make up a higher percentage of patients with treatment resistant depression. Furthermore, men and women, diagnosed with depression, experience different symptoms, suggesting there might be sex specific mechanisms in susceptibility and development of depression. Stress, being a potent trigger for various mood disorders, including depression, affects both the peripheral and central immune systems, including microglia, the resident immune cells of the brain. In this work, I investigated the impact of stress on microglia activation in the nucleus accumbens and hippocampus, brain regions associated with depression, and found sex differences in activation of microglia following variable stress. To further understand the role of microglial in development of depression we conducted a number of different experiments, pharmacologically manipulating the activation state of microglia in the nucleus accumbens in female mice. We found that shifting microglia to an anti-inflammatory state promoted social resilience in female mice after 6 days of variable stress. We further demonstrated that blocking pro-inflammatory activation of microglia did not prevent the stress effects on behavior. Surprisingly, prior pro-inflammatory activation of microglia was protective against subsequent variable stress. Taken together this work contributes to a growing number of studies demonstrating neuro-immune mechanisms associated with depression.

No description available.

No description available.

No description available.

In patients with multiple sclerosis (MS), microglia become activated due to the autoimmune inflammatory response which is directed against the central nervous system (CNS). Following the first disease relapse, microglia remain activated and do not return to a resting state during remissions. Chronically-activated microglia release inflammatory mediators that cause CNS tissue damage, and as such, MS progression has been associated with widespread, chronic microglial activation that correlates with neurodegeneration. To date, only one histone demethylase, Jmjd3, has been described to have a role in inflammation. In agreement with this, up-regulation of Jmjd3 expression was observed following microglial treatment with several pro-inflammatory stimuli, including a range of toll-like receptors ligands and cytokines, suggesting a universal role of Jmjd3 during microglial activation. Subsequent ChIP-qPCR assays revealed that Jmjd3 was recruited to the promoters of Il6, Ccl3, Ccl5 and Nos2 following activation, which, in turn, presented a decrease in their H3K27me3 levels. Using an experimental autoimmune encephalomyelitis (EAE) mouse model of MS, Jmjd3 expression was shown to be increased in activated microglia from mice in the acute and late phases of disease. Immunization with complete Freud’s adjuvant (CFA) alone, also caused microglial activation with Jmjd3 induction, indicating a CFA-mediated TLR2 and TLR4 stimulation of microglia. Further investigation, in which primary microglia were isolated from mice deficient in Jmjd3 (Jmjd3-/-), however demonstrated that the absence of Jmjd3 alone had no resultant effect on the expression of a subset of immune response and inflammation related genes, including the Jmjd3 target genes Il6, Ccl3, Ccl5 and Nos2, before or after activation. This suggested that Jmjd3 acts in concert with a repertoire of other demethylases to facilitate microglia activation, and as such was rendered redundant in this setting. Deciphering the epigenetic profile of microglia in MS and determining whether it is involved in the maintenance of chronic microglial activation in the progressive phase of the disease remains an important line of investigation, and through a clearer understanding of its role in MS pathophysiology, could lead to the development of novel therapeutic interventions in the future.