Em estudo anterior, observamos que o Met-RANTES, antagonista de receptores CCR1 e CCR5 para quimiocinas, injetado pela via endovenosa (i.v.) reduziu a resposta febril induzida pelo lipopolissacarídeo (LPS) de E. coli, demonstrando o envolvimento da quimiocina RANTES (Regulada sob ativação, expressa e secretada por células T normais) nesta resposta. Além disso, a injeção intrahipotalâmica (i.h.) da RANTES dose-dependentemente aumentou a temperatura corporal de ratos, o qual foi caracterizado como febre, pois foi acompanhada de redução da temperatura da cauda, uma resposta termorregulatória para retenção de calor. Observamos também, que a RANTES aumenta a concentração de prostaglandinas no fluido cerebroespinhal (CSF) e que a febre por ela induzida é sensível aos inibidores não-seletivos para as ciclooxigenases e seletivo para COX-2 (Machado et al., 2007). No presente estudo, aprofundamos a investigação sobre os mediadores, incluindo as prostaglandinas, envolvidos na resposta febril induzida pela RANTES. Verificamos que o paracetamol reduziu, enquanto o diclofenaco de sódio aboliu a resposta febril induzida pela RANTES. Ainda, a injeção i.h. da RANTES promoveu significativa expressão do RNAm para COX-2 no hipotálamo, confirmando ser a COX-2 a enzima responsável pela síntese de prostaglandinas envolvidas no efeito pirogênico desta quimiocina. Através da administração de corante in situ e de cortes histológicos, pode-se averiguar o trajeto da cânula bem como a profundidade alcançada pela agulha durante a injeção na área pré-óptica hipotalâmica anterior (AH/POA). Padronizamos a dose do Met-RANTES (i.h.) que não seria capaz de alterar a temperatura retal dos animais. Posteriormente, avaliou-se o efeito de diferentes doses do Met-RANTES, administrado via intrahipotalâmica, na resposta febril induzida pelo LPS ou pela RANTES. Entretanto, nas doses administradas o pré-tratamento com o antagonista não foi capaz de reduzir a febre induzida por ambos os estímulos. Contudo, o Met-RANTES (i.v.) reduziu a febre induzida pelo TNF-alfa (i.h.), reproduzindo resultados anteriores. O pré-tratamento com Met-RANTES (i.v.) não modificou a febre induzida pela injeção central de interleucina (IL)-6, fator liberador de corticotropina (CRF) e bradicinina (BK). Adicionalmente, a injeção de LPS (i.v.) ou TNF-alfa (i.h.) elevou a concentração da RANTES no tecido hipotalâmico. Antalarmina (antagonista de receptores CRF1) e alfa-helical CRF9-41 (antagonista de receptores CRF1 e CRF2) que reduziram a febre induzida pelo CRF, não alteraram a febre induzida pela administração i.h. da RANTES. O antagonista de receptores B1 (DALBK) que reduziu a segunda fase da resposta febril induzida pela BK, não foi capaz de modificar a febre induzida pela RANTES. Da mesma forma, o antagonista de receptores B2 (Hoe-140) que reduziu a resposta febril induzida pela BK durante todo o período de experimentação, não modificou a febre promovida pela RANTES. Por outro lado, verificamos que o anticorpo anti-IL-6 administrado i.h. reduziu a febre induzida pela IL-6 e pela RANTES. Ainda, a injeção de LPS (i.v.) ou RANTES (i.h.) elevou a concentração de IL-6 no CSF, mas não de IL-1 e TNF-. A RANTES promoveu ativação do fator nuclear-kB (NF-kB) e aumentou a expressão do RNAm para as citocinas IL-1beta, TNF-alfa e IL-6 no hipotálamo dos animais. O pré-tratamento com Met-RANTES reduziu, na 2,5 e 6 h, a neutrofilia induzida pelo LPS. Em síntese, nossos resultados demonstram que durante a resposta febril induzida pelo LPS, este induz a síntese de TNF-alfa o qual promove a síntese da quimiocina RANTES que, ativando os receptores CCR1 e CCR5 promove a transmigração do NF-kB do citoplasma para o núcleo e a subseqüente síntese de IL-6 e de COX-2, esta última, a responsável pela síntese de prostaglandina E2 (PGE2), um dos mediadores finais da resposta febril induzida pelo LPS. Além disso, a RANTES parece ser um mediador da resposta de fase aguda, uma vez que, promove dois sinais importantes desta resposta, febre e neutrofilia. / We showed before that Met-RANTES, CCR1 and CCR5 receptor antagonist, intravenously injected (i.v.) reduced fever induced by lipopolysaccharide (LPS, E. coli), demonstrating the involvement of RANTES (Regulated on activation, normal T cells expressed and secreted) in this response. Also, intrahypothalamic (i.h.) injection of RANTES dose-dependently increased body temperature of rats, this increase was characterized as fever, because it was accompanied of a reduction in the tail skin temperature, a thermoregulatory response for heat retention. We also verified that RANTES increased the concentration of prostaglandin (PG)E2 in the cerebrospinal fluid (CSF), which was sensible to non-selective and selective blockers to cyclooxygenase (COX)-2 (Machado et al., 2007). In the present study, it was investigated which others mediators, including prostaglandins, are involved in the RANTES-induced fever. The effect of paracetamol and sodium diclofenac on fever induced by RANTES was also investigated. Paracetamol reduced, while sodium diclofenac abolished the RANTES-induced fever. The intrahypothalamic (i.h.) RANTES injection promoted a significant COX-2 mRNA expression in the hypothalamus, confirming the role of the COX-2 enzyme in the synthesis of prostaglandin involved in the pyrogenic effect of this chemokine. Through administration of dye in situ and histological analyses, we confirmed that the injection in the preoptic area of the anterior hypothalamus (AH/POA) was correct. Subsequently, we evaluated the effect of different doses of Met-RANTES (i.h.) in the fever induced by both LPS and RANTES. Centrally injected, Met-RANTES did not modify the fever induced by LPS or RANTES. On the other hand, Met-RANTES (i.v.) reduced TNF-alpha-induced fever, but did not modify the fever induced by interleukin (IL)-6, corticotrophin releasing factor (CRF) and bradykinin (BK). Additionally, the injection of LPS (i.v.) or TNF-alpha (i.h.) increased RANTES concentration in the hypothalamus. Antalarmin (a CRF receptor 1 antagonist) and alpha-helical CRF9-41 (CRF 1 and 2 receptor antagonist) that reduced CRF-induced fever did not modify the fever induced by RANTES (i.h.). DALBK (bradykinin B1 receptor antagonist) that reduced the second phase of BK-induced fever did not modify RANTES-induced fever. In the same way, Hoe-140 (bradykinin B2 receptor antagonist) that reduced the fever induced by BK during the whole period of observation, did not modify RANTES-induced fever. On the other hand, we verified that anti-rat IL-6 antibody (i.h.) reduced the fever induced by both IL-6 and RANTES. In addition, the administration of LPS (i.v.) or RANTES (i.h.) increased the CSF IL-6 concentration, but not of IL-1 and TNF-. RANTES promoted nuclear factor-kB (NF-kB) activation and increased IL-1beta, TNF-alpha and IL-6 mRNA expression in the hypothalamus. Pretreatment of the animals with Met-RANTES reduced the LPS-induced neutrophilia. In synthesis, our results suggest that in the fever induced by LPS, RANTES induces TNF- synthesis, which promotes the synthesis of RANTES that, activating CCR1/CCR5 receptors, promotes NF-kB transmigration of cytoplasm to the nucleus and subsequent synthesis of IL-6 and COX-2. The latter, in turn, is responsible by (PGE2) synthesis, one of the final mediators of the febrile response induced by LPS. Moreover, RANTES seem to be a mediator of the acute phase response since it promoted two important signs of this response, fever and neutrophilia.

Os mecanismos que conferem resistência do Mycobacterium tuberculosis (Mtb) à destruição pelo hospedeiro, além da sua capacidade em permanecer e/ou multiplicar-se no interior das células fagocitárias são ainda pouco compreendidos. Nosso grupo de pesquisa tem contribuído para o entendimento do papel dos mediadores lipídicos, que incluem prostaglandinas (PGs) e leucotrienos (LTs) na tuberculose. PGs inibem a resposta imune celular TH1, a produção de citocinas e a fagocitose, e assim facilita a infecção. LTs estão envolvidos no recrutamento de leucócitos, e na modulação da síntese de citocinas, no aumento da fagocitose e dos mecanismos microbicidas, e assim contribui para a eliminação da micobactéria. Neste projeto, avaliamos in vivo e in vitro a produção dos mediadores lipídicos induzidos por cepas de Mtb isolados de pacientes com tuberculose ativa. Demonstramos neste trabalho que macrófagos alveolares infectados com os bacilos da cepa SV009 levam a maior produção de TNF- e nitrito, do que aqueles infectados com a cepa SV068. Em contraste, macrófagos alveolares infectados com os bacilos da cepa SV068 induzem a produção de muito mais LTB4, quando comparado aos bacilos da cepa SV009. Obtivemos maior recuperação de unidades formadoras de colônia (UFC) de macrófagos alveolares tratados com MK886 e infectados com bacilos da cepa SV068; enquanto que mais UFCs foram recuperadas após o tratamento com ácido caféico e infecção com a cepa SV009. Com relação a formação de corpúsculos lipídicos (CLs), observamos um maior número destes quando macrófagos alveolares foram infectados com bacilos da cepa SV068. Ainda, observamos diminuição de CLs quando tratados com MK886 ou ácido caféico. Os bacilos da cepa SV068 foram mais fagocitados, mas os macrófagos não foram muito eficazes na atividade microbicida dos mesmos. Nos experimentos in vivo vimos que camundongos balb/c infectados com a cepa SV068 morrem mais e o tratamento com MK886 parcialmente os protege e a mortalidade não está relacionada com a maior carga bacilar no pulmão ou baço. Houve aumento no recrutamento de neutrófilos induzido pela infecção especialmente após infecção com os bacilos da cepa SV068, sendo que o tratamento com MK886 inibe significativamente o recrutamento quando comparado à infecção com os bacilos da cepa SV009. Células mononucleares também foram recrutadas e permaneceram aumentadas até o final do período observado, sem muitas diferenças significativas quando comparamos a infecção com os isolados SV009 e SV068. A produção de nitrito também encontrou-se elevada em animais infectados com bacilos da cepa SV068. A análise histopatológica dos pulmões dos animais infectados mostrou intensa reação inflamatória com maior comprometimento do parênquima pulmonar dos camundongos infectados bacilos da cepa SV068, com intensa deposição de colágeno e multiplicação bacilar. Encontramos diferenças significativas em relação à producão de citocinas IL-6, IL-10, IL-1, IFN-, TNF- and IL-12 após infecção de 30 e 60 dias com as cepas SV009 e SV068. Também mostramos que há diferenças na produção de LTB4 e PGE2 após 30 e 60 dias de infecção com as cepas SV009 e SV068 em células do camundongos balb/c. Experimentos com animais 129 e 5LO-/- infectados com as duas cepas também foram realizados, e vimos que os animais 5LO-/- são mais suscetíveis à infecção especialmente quando infectados com a cepa SV068. Sugerimos que as cepas são diferentes, mas dependentes de um conjunto de fatores, e nossos dados sugerem que dentre estes mecanismos a produção de TNF- e também de mediadores lipídicos (LTB4 e PGE2) estão envolvidos. / The mechanisms that confer resistance to Mycobacterium tuberculosis (Mtb) for destruction by the host, in addition to its ability to retain and/or multiply within phagocytic cells are still poorly understood. Our research group has contributed to the understanding of the role of lipid mediators, including prostaglandins (PGs) and leukotrienes (LTs) in tuberculosis. PGs inhibit Th1 cell immune response, cytokine production and phagocytosis, thus facilitating the infection. LTs are involved in the leukocytes recruitment, and modulation of cytokine synthesis, phagocytosis and microbicidal mechanisms enhancement, and contribute to the elimination of the mycobacteria. In this project, we evaluated in vivo and in vitro the lipid mediators production induced by Mtb strains isolated from patients with active tuberculosis. We demonstrated in this study that alveolar macrophages infected with bacilli from SV009 strain lead to an increase of TNF- production and nitrite, than those infected with the strain SV068. In contrast, alveolar macrophages infected with bacilli from SV068 strain induced more LTB4 production when compared to SV009 infection. We obtained higher recovery colony forming units (CFU) of alveolar macrophages treated with MK886 and infected with bacilli from SV068 strain; while more CFUs were recovered after treatment with caffeic acid and infection with bacilli from SV009 strain. Regarding the lipid bodies (LBs) formation, we observed a greater number of these structures, when alveolar macrophages were infected with bacilli from SV068 strain. Still, we observed a decrease of LBs when the macrophages were treated with MK886 and caffeic acid. Bacilli from SV068 strain were more phagocytosed, but macrophages were not very effective in the microbicidal activity. In the in vivo experiments we found that mice infected with SV068 strain die more than the other and MK886 treatment partially protects the mice, besides, the mortality is not related to the higher bacterial load in the lung or spleen. There was an increase in neutrophil recruitment induced after infection, especially after infection with SV068 strain, and treatment with MK886 significantly inhibits recruitment when compared to infection with SV009 strain. Mononuclear cells were also recruited and remained increased until the end of the observed period, without many significant differences when comparing infection with SV009 and SV068 strains. The nitrite production was also found greater in animals infected with bacilli from SV068 strain. Histopathological analysis of the infected mice lungs showed an intense inflammatory reaction with greater impairment of the mice lungs when infected with bacilli from SV068 strain with an intense collagen deposition and multiplication of bacilli. We suggest that the SV068 strain is more virulent and participates of the immune response by lipid mediators dependent mechanisms.

Prostaglandin (PG) E2 is an eicosanoid derived from the polyunsaturated twenty carbon fatty acid arachidonic acid (AA). PGE2 has physiological as well as pathophysiological functions and is known to be a key mediator of inflammatory responses. Formation of PGE2 is dependent upon the activities of three specific enzymes involved in the AA cascade; phospholipase A2 (PLA2), cyclooxygenase (COX) and PGE synthase (PGEs). Although the research within this field has been intense for decades, the regulatory mechanisms concerning the PGE2 synthesising enzymes are not completely established. PGE2 was investigated in human monocytes with or without lipopolysaccharide (LPS) pre-treatment followed by stimulation with calcium ionophore, opsonised zymosan or phorbol myristate acetate (PMA). Cytosolic PLA2a (cPLA2a) was shown to be pivotal for the mobilization of AA and subsequent formation of PGE2. Although COX-1 was constitutively expressed, monocytes required expression of COX-2 protein in order to convert the mobilized AA into PGH2. The conversion of PGH2 to the final product PGE2 was to a large extent due to the action of microsomal PGEs-1 (mPGEs-1). In addition, experiments with inhibitors of extracellular signal regulated kinase and p38 activation, indicated that phosphorylation of cPLA2α was markedly advantageous for the formation of PGE2. Ellagic acid, a natural polyphenolic compound found in fruits and nuts, was shown to inhibit stimuli induced release of PGE2 in human monocytes. The effect of ellagic acid was not due to a direct effect on the activities of the enzymes but rather to inhibition of the LPS-induced protein expression of COX-2, mPGEs-1 and cPLA2a.

Conselho Nacional de Desenvolvimento Científico e Tecnológico / Methylmalonic acidemias comprehend a group of innate error of the metabolism (EIM)characterized clinically and biochemically for the tissue accumulation of acid methylmalonic (MMA)and neurological dysfunction, including seizures. The clinical experience suggests that infections precipitate metabolic crises in methylmalonic acidemic patients. Since it has been demonstrated that MMA cause seizures, and that inflammation facilitates the occurrence of seizures in some animal models, is possible that inflammatory mediators, such as the prostaglandins, also facilitate MMAinduced seizures. Ciclooxigenase (COX) is the rate-limiting enzyme in the metabolic route by which the arachidonic acid is converted to prostaglandins. COX-2 is an isoform of cicloooxigenase that is induced at sites of injury / inflammation, and that is also constitutively expressed in some tissues, such as the central nervous system (CNS). It has been suggested that prostaglandin E2 (PGE2), the main product of COX-2 in the CNS, plays an important role in some neurodegenerative diseases, including epilepsy. However, no study has evaluated whether inflammatory mediators, such as the PGE2, facilitates MMA-induced seizures, to date. Thus, in this study we investigated the role of COX-2 and of PGE2 in seizures induced by MMA (2,5 µmol/2,5 µL, i.c.v.). While PGE2 (100 ng/2 μL, i.c.v.) facilitated, the selective COX-2 inhibitor celecoxib, attenuated MMA-induced seizures, assessed by electroencephalographic recordings in the hippocampus and cerebral cortex of rats. The ´protective effect of celecoxib against MMA-induced seizures was prevented by the PGE2. The results of this study support a facilitatory role for COX-2/PGE2 pathway in the MMA-induced seizures. / A acidemia metilmalônica é um erro inato do metabolismo (EIM) caracterizado bioquimicamente e clinicamente pelo acúmulo tecidual de ácido metilmalônico (MMA) e disfunção neurológica, que inclui convulsões. A experiência clínica sugere que infecções precipitam crises metabólicas em pacientes metilmalonicacidêmicos. À medida em que foi demonstrado que o MMA causa convulsões, e que a inflamação pode contribuir para a ocorrência de convulsões em vários modelos animais, é possível que mediadores inflamatórios, como as prostaglandinas, facilitem as convulsões induzidas por MMA. A ciclooxigenase (COX) é a enzima marca-passo na rota metabólica pela qual o ácido araquidônico é convertido em prostaglandinas, e a COX-2 uma isoforma da cicloooxigenase, é induzida em sítios de lesão/inflamação, e também se expressa constituivamente em alguns tecidos, como o sistema nervoso central (SNC). Tem sido sugerido que a prostaglandina E2 (PGE2), principal produto da via COX-2 no SNC, tenha um papel importante em várias doenças neurodegenerativas, incluindo epilepsia. Contudo, até a presente data nenhum estudo avaliou se mediadores inflamatórios, como a PGE2, facilitam as convulsões induzidas por MMA. Assim, neste estudo investigamos o papel da COX-2 e da PGE2 nas convulsões induzidas por MMA (2,5 µmol/2,5 µL, i.c.v.). Verificamos que a PGE2 (100 ng/2 μL, i.c.v.) facilita as convulsões induzidas por este ácido orgânico. Além disso, verificamos que o celecoxibe, um inibidor seletivo da COX-2, na dose de 2 mg/kg (v.o.), atenuou as convulsões comportamentais e eletrográficas induzidas por MMA no hipocampo e córtex cerebral de ratos. O efeito protetor do celecoxibe contra as convulsões induzidas por MMA foi revertido pela administração i.c.v. de prostaglandina E2. O conjunto de dados deste estudo suporta um papel facilitatório para a via COX-2/PGE2 nas convulsões induzidas por MMA.

Prostaglandin E2 (PGE2), a metabolite of arachidonic acid, plays a role in water and sodium reabsorption in the collecting duct of the kidney. The collecting duct is responsible for the fine tuning of water and electrolytes. Only a small fraction of the filtered water and sodium is reabsorbed in the collecting duct, a fraction crucial to the regulation of water and electrolyte balance. This current study addresses the role of EP1, one of four PGE2 receptors, in the collecting duct. It is well documented that PGE2 inhibits sodium and water reabsorption in the collecting duct, however the exact mechanism is still debated. To determine whether the EP1 receptor mitigates AngII renal effects, an in vivo study was performed with EP1-/- mice. Global EP1-/- knockout mice were crossed with a renin overexpressing mouse line (herein denoted as “Ren”) and subjected to a high salt (HS) and low salt (LS) diet. Ren mice displayed an 11mmHg increase in systolic blood pressure (BP) on a HS diet and a decrease in BP of 14mmHg on a LS diet compared to the normal salt (NS) diet. Ren EP1-/- mice did not display a significant increase or decrease in BP on a HS or LS diet. On a LS diet, Ren EP1-/- displayed a drop in urine osmolarity (1641 mOsm/ kgH2O) vs. wild type (WT) mice (2107 mOsm/ kgH2O), consistent with increased sodium reabsorption. Narrowing in on the collecting duct, Ren EP1-/- mice had enhanced αENaC levels compared to Ren mice. In ex vivo microperfusion experiments, EP1-/- tubules show no response to PGE2 in the presence of AVP, whereas PGE2 inhibits AVP induced water reabsorption in WT mice. An increase in αENaC membrane accumulation due to EP1 gene ablation results in increased sodium reabsorption subsequently leading to a rise in BP. This contributes to the lack of salt sensitivity in EP1-/- mice. Overall, the EP1 receptor in the collecting duct represents a potential therapeutic target for the treatment of hypertension.

The incidence of chronic kidney disease (CKD) is increasing. CKD is characterized by a gradual decrease in renal function leading to end stage renal disease (ESRD). Damage to the glomerular podocytes, is one of the first hallmarks of CKD. We hypothesized that podocyte prostaglandin E2 (PGE2) receptors contribute to the progression of glomerular injury in models of CKD. To test this hypothesis, transgenic mice were generated with either podocyte-specific overexpression or deletion of the PGE2 EP4 receptor (EP4pod+and EP4pod-/- respectively). Mice were next tested in the 5/6 nephrectomy (5/6 Nx) or angiotensin II (Ang II) models of CKD. These studies revealed increased proteinuria and decreased survival for EP4pod+ mice while EP4pod-/- mice were protected against the development of glomerular injury. Furthermore, our findings were supported by in vitro studies using cultured mouse podocytes where an adhesion defect was uncovered for cells overexpressing the EP4 receptor. Additionally, our investigations have demonstrated a novel synergy between angiotensin II AT1 receptors and prostaglandin E2 EP4 receptors. This was revealed by in vitro studies using isolated mouse glomeruli. There we were able to show that Ang II stimulation leads to increased expression of cyclooxygenase 2 (COX-2), the enzyme responsible for synthesis of PGE2, in a p38 mitogen activated protein kinase (MAPK) dependent fashion. Moreover increased PGE2 synthesis was measured in response to Ang II stimulation. We confirmed the presence of this synergy in our cultured mouse podocytes and showed an adhesion defect in response to Ang II stimulation which was COX-2 and EP4 dependent. These findings suggest that Ang II AT1 receptors and PGE2 EP4 receptors act in concert to exacerbate glomerulopathies. Studies using mice with either podocyte-specific overexpression of a dominant negative p38 MAPK or mice with global deletion of the EP1 receptor did not provide conclusive results as to their respective signaling involvement in podocyte injury. Altogether our findings provide novel insight for podocyte PGE2 EP4 and Ang II AT1 receptor signaling in models of CKD. These studies provide novel avenues for pursuing therapeutic interventions for individuals with progressive kidney disease.

Ziel dieser Arbeit war die Entwicklung optimaler DCs für die Generierung von therapeutischen Tumorvakzinen. Neben den essenziellen Eigenschaften der DCs wie der Migrationsfähigkeit, der Hochregulation kostimulatorischer Moleküle sowie der Zytokinausschüttung, ist auch die optimale Aufnahme von Tumor-spezifischen Antigenen und deren Präsentation besonders wichtig, um eine effiziente Immunantwort zur ermöglichen. Wichtigstes Ziel war es über einen optimalen Ausreifungscocktail der DCS eine effektive Antwort der zytotoxischen CD8\(^+\)-Zellen zu erzielen. Dies geschieht im Rahmen der Präsentation von z.B. exogenen Antigenen wie in unserem Falle eines CMV-Proteins über den Weg der sogenannten Kreuzpräsentation. Hierzu wurde im Rahmen dieser Arbeit der in unserer Klinik etablierte zytokinbasierte Ausreifungscocktail (IL-1β, TNFα) mit einem Ausreifungscocktail, welcher Toll-like-Rezeptor-Agonisten mit PGE\(_2\) kombiniert, verglichen. Wir konnten erstmals zeigen, dass PGE\(_2\) nicht nur einen Einfluss auf die Ausreifung, Zytokinproduktion und somit Migrationsfähigkeit der DCs hat wie in der Literatur beschrieben, sondern auch auf die Kreuzpräsentationsfähigkeit exogener Antigene. Das Weglassen von PGE\(_2\) im Cocktail 2 führte zu einer signifikant besseren Kreuzpräsentationsfähigkeit. Somit lässt sich durch unsere Experimente auf eine hemmende Wirkung von PGE\(_2\) auf die T-Zell-Aktivierung über die Kreuzpräsentation schließen. Als mögliche Schlussfolgerung unserer Experimente sollte bei der Arbeit mit Antigenen, welche über Kreuzpräsentation eine T-Zell-Antwort auslösen (Proteine, Tumorlysat, long-peptides) auf die Zugabe von PGE\(_2\) im Ausreifungscocktail verzichtet werden. / The aim of this work was to develop optimal dentritic cells (DCs) for the generation of therapeutic tumor vaccines. Besides the essential properties of DCs such as migration ability, upregulation of costimulatory molecules as well as cytokine release, the optimal uptake of tumor-specific antigens and their presentation is also particularly important to enable an efficient immune response. The most important goal was to achieve an effective response of cytotoxic CD8\(^+\) cells via an optimal maturation cocktail for DCs. This is done in the context of the presentation of e.g. exogenous antigens like in our case a CMV protein via cross-presentation. For this purpose, we compared the cytokine-based maturation cocktail (IL-1β, TNFα) established in our clinic with a maturation cocktail combining Toll-like receptor agonists with prostaglandin E2 (PGE\(_2\)). We were able to show for the first time that PGE\(_2\) not only has an impact on maturation, cytokine production and thus migratory ability of DCs as described in the literature, but also on the cross-presentation ability of exogenous antigens. Elimination of PGE\(_2\) in cocktail 2 resulted in significantly better cross-presentation ability. Thus, our experiments suggest an inhibitory effect of PGE\(_2\) on T-cell activation via cross-presentation. As a possible conclusion of our experiments, the addition of PGE\(_2\) in the maturation cocktail should be avoided when working with antigens that trigger a T cell response via cross-presentation (proteins, tumor lysate, long-peptides).

no / Erythema occurs in human skin following excessive exposure to ultraviolet radiation (UVR), and this is in part mediated by the vasodilator prostaglandin E2 (PGE2). While keratinocytes are a major source of this pro-inflammatory eicosanoid, epidermal melanocytes (EM) also express some of the cellular machinery required for PGE2 production. The primary aim of this study is to determine whether EM can produce PGE2 and so potentially also contribute to UVR-induced skin inflammation. Furthermore, we investigate the likely pathway by which this PGE2 production is achieved and investigate whether PGE2 production by EM is correlated with melanogenic capacity. Primary cultures of EM were established from nine normal healthy individuals with skin phototype-1 (n=4) and 4 (n=5), and PGE2 production and melanogenic status were assessed. EM produced PGE2 under baseline conditions and this was increased further upon stimulation with arachidonic acid. Moreover, EM expressed cytoplasmic phospholipase A2, cyclooxygenase-1 and cytoplasmic prostaglandin E synthase. However, no EM culture expressed cyclooxygenase-2 under baseline conditions or following arachidonic acid, UVB- or H2O2 treatments. PGE2 production in response to UVB was highly variable in EM cultures derived from different donors but when pooled for skin phototype exhibited a positive correlation only with SPT-1 derived EM. Interestingly, PGE2 production by EM in response to UVB showed no correlation with baseline levels of melanin, tyrosinase expression/activity or tyrosinase-related protein-1 expression. However, there was an apparent negative correlation with baseline expression of dopachrome tautomerase (DCT), a melanogenic enzyme with reported anti-oxidant potential. These findings suggest that EM have the potential to contribute to UVR-induced erythema via PGE2 production, but that this response may be more related to oxidative stress than to their melanogenesis status. / The Wellcome Trust

No description available.

Interleukin-27 (p28/EBI3) is an immunomodulatory cytokine expressed by activated antigen presenting cells. Although first discovered to be involved in Th1 cell differentiation, further studies demonstrated the immunosuppressive functions of IL-27 including inhibition of Th2 and Th17 differentiation, development of a tolerogenic phenotype in dendritic cells (DC), and promoting type 1 regulatory T cells (Tr1). The anti-inflammatory effects of IL-27 have been demonstrated in vivo in murine models of parasitic infections and autoimmune diseases. Despite the prevalence of studies detailing the induction of IL-27 expression and the role of IL-27 in Tr1 differentiation, little is known about factors that negatively regulate IL-27 expression and Tr1 differentiation. Prostaglandin E2 (PGE2), a lipid mediator abundant at inflammatory sites, was shown to act as a proinflammatory agent in models of inflammatory/autoimmune diseases primarily by promoting CD4 Th1/Th17 differentiation. Here we describe a novel proinflammatory mechanism for PGE2 through the inhibition of IL-27 production in conventional dendritic cells (cDC) and the inhibition of Tr1 differentiation. PGE2 inhibits IL-27 production in bone marrow-derived DC and macrophages, as well as in splenic cDC, through EP2/EP4 receptors, induction of cAMP, and downregulation of IRF1 expression and binding to the p28 IL-27 ISRE site. The inhibitory effect of PGE2 on p28 and irf1 expression does not involve endogenous IFN-β, STAT1 or STAT2, and inhibition of IL-27 does not appear to be mediated through PKA, EPAC, PI3K, or MAPKs. We observed similar inhibition of p28 expression in vivo in splenic DC following administration of dimethyl PGE2 in conjunction with LPS. In addition to the inhibition of IL-27 production in APCs, PGE2 also directly affects Tr1 differentiation by reducing IL-27-induced CD4+CD49b+LAG-3+Foxp3- Tr1 cells and IL-10 production. The inhibitory effect is mediated by EP4 and induction of cAMP in differentiating CD4 T cells. IL-27-induced Tr1 differentiation and function depends primarily on the sustained expression of c-Maf in addition to AhR and Blimp-1. PGE2 significantly reduced expression of c-Maf without affecting AhR and only marginally reducing Egr-2/Blimp-1 expression. The effects of PGE2 on Tr1 cells are independent of STAT1/STAT3 signaling and of IL-21 signaling. In addition, the effect of PGE2 on CD4+CD49b+LAG-3+ Tr1 differentiation was not associated with either induction of Foxp3 or IL-17 production, suggesting a lack of transdifferentiation into Foxp3+ Treg or effector Th17 cells. The effects of PGE2 on both IL-27 production and IL-27-induced Tr1 differentiation represent novel proinflammatory mechanisms of PGE2. / Microbiology and Immunology