O escorpião Tityus serrulatus é considerado uma das espécies mais perigosas para os seres humanos no Brasil, e sua peçonha induz resposta inflamatória local e sistêmica. Neste projeto, tivemos como objetivo estudar a produção de mediadores inflamatórios, as vias de ativação celular e os receptores da imunidade inata responsáveis pelo reconhecimento da peçonha do escorpião T. serrulatus (TsV), bem como de suas toxinas. Nós demonstramos que TsV, e suas toxinas Ts1 e Ts6 induzem a produção de NO, IL-6 e TNF-? por células J774.1, as quais podem ser potencializadas pela presença de LPS. No entanto, Ts2 apresenta atividade anti-inflamatória por induzir produção de IL-10 e inibe a liberação de NO, IL-6 e TNF-?, induzida pelo LPS. Mostramos ainda que Ts2 ou Ts6 isoladas do TsV, além das citocinas, induzem a produção dos mediadores lipídicos (LTB4 e PGE2), e estes contribuem para o recrutamento de leucócitos para a cavidade peritoneal. Em conjunto, os nossos dados demonstraram que Ts2 e Ts6 induzem inflamação por mecanismos dependentes da produção de citocinas e mediadores lipídicos, e que Ts2 pode desempenhar papel regulador da resposta. No entanto, os mecanismos responsáveis pelo reconhecimento da peçonha e indução da liberação de mediadores inflamatórios por células de mamíferos, são desconhecidos. Assim, dando continuidade aos nossos estudos, demonstramos que os receptores TLR2, TLR4 e CD14 reconhecem TsV, e medeiam a produção de citocinas e mediadores lipídicos. Além disso, nós demonstramos que TsV ativa NF-?B dependente de MyD88, e o fator c-Jun, independente de MyD88. Semelhante ao TsV, a sua toxina majoritária, Ts1, induz a fosforilação de NF-?B dependente de MyD88, via reconhecimento por TLR2 e TLR4, enquanto a ativação c-Jun é via TLR4, mas independente de MyD88. Dentro deste contexto, nós propusemos o termo Padrões Moleculares Associados à Venenos (VAMP) para se referir às moléculas que são introduzidas no hospedeiro por picadas e são reconhecidas por receptores de reconhecimento padrão (PRRs), resultando em inflamação. Demonstramos ainda, a formação de corpúsculos lipídios (CLs) e a geração de eicosanóides, após o reconhecimento do TsV por TLR2 e TLR4. Nossos dados mostraram que a formação de eicosanóides se correlaciona com a formação dos CLs, que por sua vez são dependentes de TLR2 e TLR4, e da ativação de PPAR?, sugerindo que este receptor nuclear pode modular a produção de citocinas pró-inflamatórias. Assim, concluímos que PPAR? pode ser um candidato-alvo atrativo para novas estratégias terapêuticas para prevenção dos efeitos deletérios resultantes da intensa liberação sistêmica de mediadores inflamatórios após envenenamento. / Tityus serrulatus is the scorpion considered one of the most dangerous species to humans in Brazil, which venom induces local and systemic inflammatory response. In this project, we aimed to study In this project, we aimed to study the inflammatory mediators production, cell activation and receptors of innate immunity responsible for recognition of the venom of the scorpion T. serrulatus (TsV) as well as their toxins. We have demonstrated that TsV and their toxins Ts1 and Ts6 induce NO, IL-6 and TNF-? production in J774.1 cells, which may be potentiated by presence of LPS. However, Ts2 exhibits anti-inflammatory activity due induction of IL-10 production and inhibits the release of NO, IL- 6 and TNF-? induced by LPS. We also show that Ts2 or Ts6 isolated of TsV, besides of the cytokines, induce the production of lipid mediators (LTB4 and PGE2), and these mediators contribute to leukocytes recruitment into the peritoneal cavity. Taken together, our data demonstrated that Ts2 and Ts6 induce inflammation by mechanisms dependent on the production of cytokines and lipid mediators, and that Ts2 may play regulatory role on the cell response. Furthermore, continuing our studies, we demonstrated that TLR2, TLR4 and CD14 receptors recognize TsV, mediating cytokines and lipid mediators production. We also showed TsV MyD88- dependent activation of NF-?B, and a MyD88-independent activation of the factor c-Jun. Similar to TsV, the majority toxin Ts1 induces MyD88-dependent phosphorylation of NF-kB via TLR2 and TLR4 recognition, while the c-Jun activation is through TLR4 recognition, but independent of MyD88. Within this context, we propose the term Venom-Associated Molecular Pattern (VAMP), to refer molecules that are introduced into the host by st ings and recognized by PRRs, resulting in inflammation. Finally, we investigated the formation of lipid bodies (LBs) and generation of eicosanoids, through TsV recognition by TLR2 and TLR4. Our data showed that eicosanoid production correlates with the LBs formation, which are dependent on TLR2, TLR4 and PPAR? activation, suggesting that this nuclear receptor can modulate cytokines inflammatory. Thus, we suggest that PPAR? may be an attractive candidate target for novel therapeutic strategies to prevent the deleterious effects of intense systemic release of inflammatory mediators after envenomation.

Im ersten Teil dieser Dissertation wurde der Einfluss des mitochondrialen Proteins TCAIM (T cell activation inhibitor, mitochondrial) auf die T-Zellaktivierung untersucht. Hierzu wurde eine transgene Mauslinie mit einem T-zellspezifischen knock-in (KI) von Tcaim in den Rosa26 Lokus generiert. Die Tcaim-Überexpression beeinflusste die Fission und Umverteilung von Mitochondrien und reduzierte die T-Zellrezeptor (TZR)-induzierte Bildung mitochondrialer, radikaler Sauerstoffspezies. In vitro stimulierte CD4+ Tcaim KI T-Zellen zeigten eine geringere Aktivierung, Proliferation und IL-2 Sekretion als Kontrollzellen. T-Zellen aus Tcaim KI Mäusen, die in Rag-1 knock-out Mäuse transferiert wurden, waren nicht fähig ein allogenes Haut-Transplantat abzustoßen und behielten einen naiven Phänotyp. Diese Ergebnisse zeigen, dass TCAIM als mitochondriales Protein wichtige Schritte in der Zellaktivierung und der Bildung von Gedächtnis-T-Zellen beeinflusst. Der zweite Teil der Dissertation beschäftigte sich mit dem Einfluss der CD44-Oberflächenexpression auf die Differenzierung von T-Helfer (TH)-Zellen. Eine hohe CD44-Expression unterscheidet Effektor- von naiven T-Zellen. Durch die allogene Stimulation von CD4+ T-Zellen bildeten sich drei verschiedene Populationen: CD44+, CD44++ und CD44+++. Sowohl in vitro als auch in vivo generierte alloreaktive TH17-Zellen wurden in der CD44+++ Population, TH1-Zellen hingegen in der CD44++ Population, detektiert. Es wurde beschrieben, dass sowohl eine geringe TZR- als auch eine geringe CD28-Stimulation eher die Bildung von TH17- als TH1-Zellen unterstützen. Unter genau diesen Bedingungen kann CD44 als kostimulatorisches Molekül die Signaltransduktion verstärken. Tatsächlich zeigten allogenreaktive CD44+++ TH-Zellen eine höhere ZAP-70-Phosphorylierung als CD44++ TH-Zellen. Diese Ergebnisse unterstützen die Annahme, dass CD44 durch die Verstärkung der Signaltransduktion die TH17-Differenzierung fördern kann. / Within the first part of this thesis, the influence of the mitochondrial Protein TCAIM (T cell activation inhibitor, mitochondrial) on T cell activation was investigated. Tcaim expression correlated negatively with the rejection of allografts and it is down-regulated during T cell activation. To study effects of TCAIM during T cell activation, we generated a T cell-specific mouse strain with a Tcaim knock-in (KI) targeted to the Rosa26 locus. Tcaim overexpression changed the mitochondrial morphology and reduced the T cell receptor (TCR)-induced mitochondrial reactive oxygen species production. In vitro activation of Tcaim KI CD4+ T cells resulted in a decreased activation, proliferation and cytokine release. Importantly, Rag-1 knock-out mice, reconstituted with Tcaim KI T cells, tolerated allogeneic skin grafts. Thus, by regulating TCR-induced mitochondrial distribution and ROS production, TCAIM controls important steps during T cell activation and memory formation. The second part dealt with the influence of CD44 surface expression level for T helper cell (Th cell) differentiation. By association with lymphocyte-specific protein kinase (LCK) it can enhance T cell signaling. Allogeneic stimulation of CD4+ T cells resulted in the formation of three distinguishable populations: CD44+, CD44++ and CD44+++. In vitro and in vivo generated allo-reactive TH17 cells were mainly CD44+++. This is in contrast to TH1 cells which were dominantly CD44++. Titration experiments revealed that low TCR- and co-stimulation supports TH17 rather than TH1 development. Under exactly these conditions it was reported that CD44 can act as co-stimulatory molecule and replace CD28. Indeed, CD44+++CD4+ T cells contained already more phosphorylated ZAP-70 as compared to CD44++ cells. Our results support the notion that CD44 enhances TCR signaling strength by delivering LCK, which is required to support TH17 development.

Die initialen immunologischen Prozesse, die zur Entwicklung autoimmuner Lebererkrankungen führen, sind weitgehend unbekannt. Deshalb wurden in dieser Arbeit die Antigenpräsentation, die Migration sowie der Phänotyp in vivo aktivierter CD8 T-Zellen in der Leber anhand eines Mausmodells der autoimmunen Hepatitis untersucht. Es konnte gezeigt werden, dass hepatische dendritische Zellen an der Entstehung von CD8 Effektor-T-Zellen und an der Inflammation der Leber beteiligt sind. Kupffer-Zellen dagegen nehmen im autoimmunen Kontext in der Leber eine tolerogene Funktion ein. Die in vivo in der Leber aktivierten CD8 T-Zellen zeigten spezifische Oberflächenmarker und ein ungewöhnliches Migrationsverhalten. So wurde zum einen mit Neuropilin-1 ein weitgehend unbekannter Oberflächenmarker identifiziert, zum anderen spricht die Expression von bekannten Markern, die den Aktivierungsstatus der CD8 T-Zellen definieren, für einen hybriden Phänotyp. Sie besitzen sowohl Charakteristika von naiven CD8 T-Zellen als auch von Effektorzellen, eine Eigenschaft, die auch bei zentralen Gedächtniszellen gefunden wird. In der Leber aktivierte CD8 T-Zellen können nicht nur proinflammatorische Zytokine ausschütten und somit eine Inflammation in der Leber auslösen, sondern sind außerdem in der Lage durch Lymphknoten zu zirkulieren. Dagegen ist ihnen der Zugang zum Darm verwehrt, womit eine direkte regulatorische Funktion im Darm ausgeschlossen werden kann. Obwohl auf in der Leber aktivierten CD8 T-Zellen spezifische Adhäsionsmoleküle identifiziert wurden, existiert keine exklusive gewebespezifische Migration in die Leber, wie sie etwa für im Darm aktivierte CD8 T-Zellen nachgewiesen wurde. Im darmassoziierten lymphatischen Gewebe aktivierte CD8 T-Zellen akkumulieren in der Leber und tragen möglicherweise zur Schädigung der Leber im Rahmen chronisch entzündlicher Darmerkrankungen bei. Diese Arbeit trägt somit zum besseren Verständnis der Entstehung autoimmuner Prozesse in der Leber bei. / Initial immunological processes leading to autoimmune liver diseases are largely unknown. Therefore this thesis analyzed the antigen presentation, the migration as well as the phenotype of in vivo activated CD8 T cells in the liver by employing a mouse model for autoimmune hepatitis. It was shown that hepatic dendritic cells are effective antigen-presenting cells, which contribute to the induction of functional effector CD8 T cells in the liver and hepatitis. In contrast, Kupffer cells have a tolerogenic role during autoimmune processes in the liver. CD8 T cells that were in vivo activated in the liver display specific surface markers and unusual migration patterns. On the one hand an unusual surface molecule Neuropilin-1 was identified, on the other hand expression of well-known markers defining the activation-status of CD8 T cells suggests a hybrid phenotype. They reflect aspects of naive and effector T cells, characteristics also found on central memory T cells. Liver-primed CD8 T cells do not only produce pro-inflammatory cytokines leading to hepatitis, but they also retain their ability to circulate through lymph nodes. However, they have no access to the gut, which suggests that a direct regulatory function in the gut can be excluded. Although specific adhesion molecules on CD8 T cells activated in the liver were identified, no exclusive tissue-specific migration into the liver exists, as was shown for CD8 T cells primed in the gut. CD8 T cells activated in the gut-associated lymphoid tissue accumulate in the liver, in principle enabling them to induce liver pathology in the context of inflammatory bowel disease. Thus, the here described findings contribute to the understanding of initial immunological processes in autoimmune liver diseases.

Glukokortikoide (GK) zählen zu den effizientesten Medikamenten bei der Behandlung akuter und chronischer Entzündungskrankheiten. Ihr Einsatz ist häufig durch das Auftreten zahlreicher und teilweise irreversibler Nebenwirkungen beeinträchtigt. Aus diesem Grund wurden neue Glukokortikoid-Rezeptor-Liganden, wie die nicht-steroidalen selektiven Glukokortikoid-Rezeptor-Agonisten (SEGRAs), die eine potente anti-entzündliche Wirkung bei gleichzeitig vermindertem Nebenwirkungspotential aufweisen sollen, entwickelt. Im Rahmen der vorliegenden Arbeit wurde die SEGRA-Substanz BAY1155975 hinsichtlich ihrer hemmenden Wirkung auf die CD28-kostimulierte Aktivierung primärer humaner T-Zellpopulationen mit der von klassischen GK, wie z.B. Prednisolon, verglichen. In humanen Gedächtnis/Effektor- CD4+ T-Zellen wies die höchste Konzentration von BAY1155975 im Vergleich zu Prednisolon eine statistisch signifikant größere Hemmung der CD28-kostimulierten Sekretion von Effektorzytokinen (IFN-gamma, TNF-alpha, IL17 und IL22) auf. Proliferation, Apoptose und die Expression verschiedener Aktivierungsmarker wurden dagegen durch BAY1155975 und Prednisolon gleichermaßen reguliert. Es wird eine stärkere Hemmung des Kalzium-Kalzineurin-NFAT Signalweges durch BAY1155975 in diesen Zellen vermutet. In vivo zeigten BAY1155975 und Prednisolon eine ähnlich starke Hemmung der T-Zell-vermittelten Hautentzündung im DNFB-induzierten Kontaktallergiemodell in Mäusen, wenn die Behandlung der Mäuse mit den Substanzen vor dem Challenge erfolgte. Bei einer Substanzbehandlung der Mäuse während der Sensibilisierung wurde die Hautentzündung dagegen deutlich stärker durch BAY1155975 als durch Prednisolon gehemmt. Zusammenfassend geben die Ergebnisse dieser Arbeit einen Hinweis auf eine stärkere Hemmung der T-Zellsensibilisierung und der Effektorzytokinsekretion durch die SEGRA Substanz BAY1155975 im Vergleich zum klassischen GK Prednisolon. / Glucocorticoids (GCs) are the most effective therapeutic agents for the treatment of acute and chronic inflammatory diseases. Their use is often accompanied with numerous and sometimes irreversible side-effects. Therefore, new glucocorticoid receptor (GR) ligands with should have potent anti inflammatory efficacy but a reduced side-effect profile have been developed. Non steroidal selective glucocorticoid receptor agonists (SEGRAs) represent a new class of GR ligands with an improved therapeutic index. In this study, we compared the SEGRA, BAY1155975, and the classical GC, prednisolone, regarding their suppressive effect on CD28-costimulated activation of human primary T cell subpopulations. In human memory/effector CD4+ T cells, BAY1155975 at the highest concentration exhibited a significantly stronger inhibition of CD28-costimulated effector cytokine secretion (IFN-gamma, TNF-alpha, IL17 and IL22) in comparison to prednisolone. Interestingly, proliferation, apoptosis and expression of activation markers were similarly regulated by BAY1155975 and prednisolone. Further studies on different signal transduction pathways suggested that BAY1155975 stronger inhibited the calcium-calcineurin-NFAT pathway than prednisolone in these cells. In vivo BAY1155975 and prednisolone showed comparable efficacy in inhibition of T cell dependent skin inflammation in DNFB-induced contact hypersensitivity models in mice, when mice were treated before hapten challenge. In contrast, when mice were treated around hapten sensitization markedly stronger inhibition of skin inflammation was observed for BAY1155975 than prednisolone. In summary, the data of this study give evidence for a stronger inhibition of T cell sensitization and effector cytokine secretion by the SEGRA, BAY1155975, in comparison to the classical GC, prednisolone.

Das Ergebnis einer T-Zellmediierten Immunantwort ist von der Signalvermittlung durch kostimulatorische Moleküle abhängig. Diese kostimulatorischen Moleküle sind - neben dem spezifischen Antigen - notwendig für eine vollständige T-Zellaktivierung, die es der T-Zelle erlaubt zu proliferieren, neue Oberflächenantigene zu exprimieren und Zytokine zu sezernieren. Ohne das kostimulatorische Signal wird die T-Zelle anerg oder sogar apoptotisch, eine effektive Immunantwort ist dann nicht möglich. Die vorliegende Dissertationsschrift enthält die initiale Beschreibung eines neuen kostimulatorischen Moleküls, eine umfangreiche Charakterisierung seiner Expression in vitro, sowie seiner Funktion. Das Molekül "ICOS" (ICOS steht für inducible costimulator) ist ein T-Zellspezifisches Molekül und weist eine große Homologie zu dem Prototyp eines kostimulatorischen Moleküls, dem CD28 Molekül auf. Die Experimente, die zur Charakterisierung der Induktionsbedingungen des ICOS Moleküls durchgeführt wurden, zeigen, daß die Expression des ICOS Moleküls sehr schnell nach T-Zellaktivierung induziert wird, die Expressionsstärke innerhalb von Stunden stark heraufreguliert wird und die Expression lange (mindestens 96h) auf der T-Zelloberfläche zu detektieren ist. Ein Vergleich mit anderen aktivierungsabhängigen T-Zelloberflächenmolekülen zeigt eine ICOS-spezifische Zeitkinetik der Expression, die durch verschiedene T-Zellstimuli zu induzieren ist. Eine optimale Expression des Moleküls ist zwei-signalabhängig und durch Cyclosporin A blockierbar. Bezüglich der Funktion von ICOS wurde die Wirkung der Kostimulation via ICOS auf eine Reihe von kritischen Parametern der T-Zellaktivierung analysiert. Durch die Kostimulation mit einem ICOS-spezifischen Antikörper wird konzentrationsabhängig die T-Zellproliferation induziert, unabhängig davon, ob das ersten Signal via CD3 oder via einen löslichen Stimulus, wie PHA erfolgte. Die ICOS Kostimulation bewirkt die Hochregulation von typischen T-Zellaktivierungsantigenen und sie induziert oder verstärkt die Sekretion zahlreicher Lymphokine. Die verstärkende Wirkung auf alle diese Parameter der T-Zellaktivierung führt schließlich dazu, daß die über ICOS kostimulierten Zellen in der Lage sind T-Zellhilfe für B-Zellen zu leisten, so daß diese Immunglobuline sezernieren. Wurde versucht, die direkte Interaktion des ICOS Moleküls mit seinem potentiellen Liganden bei der T-Zellinduzierten Immunglobulinsekretion durch den mAk F44 zu blockieren, so zeigte sich kein Effekt. Bei Langzeitkostimulation via ICOS zeigte sich allerdings, daß die Kostimulation durch das ICOS Molekül auch in der Lage ist, die T-Zellaktivierung negativ zu beeinflussen: Die Langzeitstimulation via ICOS erzeugt eine deutliche Proliferationsdepression und einen Viabilitätsverlust der T-Zellen. Insgesamt lassen diese Ergebnisse den vorsichtigen Schluß zu, daß das ICOS Molekül eine wichtige Rolle an der Schnittstelle zwischen Expansion und Effektorfunktion einerseits und Depression der T-Zellen andererseits spielt. / The outcome of T-cell resonses after T-cell encounter with specific antigens is modulated by co-stimulatory signals, which are required for both lymphocyte activation and development of adaptive immunity. Here I report the initial characterization of a novel co-stimulatory molecule which enhances all basic T-cell functions and displays a unique induction and expression pattern. ICOS (for inducible co-stimulator) is a T-cellspecific activation antigen with high homology to CD28, the prototype of a co-stimulatory molecule. Analysis of induction requirements for ICOS expression revealed a two-signal dependency and cyclosporine A sensitivity. ICOS' expression kinetics are unique when compared with other early T-cell activation antigens. ICOS is induced very quickly on the T-cell surface and is rapidly upregulated following T-cell activation. The surface expression of ICOS is surprisingly prolonged - lasting at least 96 hours - considering its rapid induction kinetics. Stimulation via an ICOS-specific monoclonal antibody enhances all basic T-cell functions such as proliferation, upregulation of molecules that medicate cell-cell interaction, secretion of lymphokines and effective help for antibody secreting B-cells. Costimulation via ICOS is effective regardless of the route of action of the first signal (immobilized or soluble). Blockade of the ICOS interaction with its presumed ligand on B-cells does not inhibit immunoglobulin production by these B-cells, though. Finally long-term stimulation experiments reveal a possible negative role for ICOS in regulating T-cell responses. These results indicate that ICOS is a major regulator of the adaptive immune system determining the healthy balance of negative and positive signaling during T-cell activation and differentiation.

The focus of this work was to functionally characterize the CD300e receptor expressed in human monocytes and myeloid dendritic cells and investigate the implications that receptor engagement has on their biology. We provide evidence formally supporting that CD300e functions as an activating receptor capable of regulating the innate immune response by triggering various pro- inflammatory functions including intracellular calcium mobilization, superoxide anion production, pro-inflammatory cytokine release and up-regulation of co-stimulatory molecules in myeloid cells. We also report that ligation of CD300e on the surface of monocytes results in their differentiation to functional MΦ2-like macrophages by an autocrine mechanism that involves M-CSF and its receptor (CD115). / L'objectiu d'aquest treball ha estat caracteritzar funcionalment el receptor CD300e expressat en monòcits i cèl·lules dendrítiques mieloides humanes, així com investigar les implicacions que l'activació d'aquest receptor pot tenir en la seva biologia. Demostrem formalment que el receptor CD300e funciona com un receptor activador capaç de regular la resposta immune innata activant diverses funcions proinflamatòries, incloent la mobilització de calci intracel·lular, la producció d'anió superòxid, la secreció de citocines proinflamatòries i la inducció de molècules coestimuladores en cèl·lules mieloides. També descrivim que l'activació del receptor CD300e a la superfície dels monòcits provoca la seva diferenciació cap a macròfags funcionals del tipus MΦ2 gràcies a un mecanisme autocrí que funciona a través del M-CSF i el seu receptor (CD115).

Les voies de signalisation des MAP kinases (MAPK) conventionnelles jouent des rôles essentiels pendant le développement des lymphocytes T (LT) ainsi que lors de leur activation suite à la reconnaissance antigénique. En raison de ses différences structurelles ainsi que de son mode de régulation, ERK3 fait partie des MAPK dites non-conventionnelles. Encore aujourd’hui, les événements menant à l’activation de ERK3, ses substrats ou partenaires ainsi que sa fonction physiologique demeurent peu caractérisés. Nous avons entrepris dans cette thèse d’étudier le rôle de ERK3 lors du développement et de l’activation des LT en utilisant un modèle de souris déficient pour l’expression de ERK3. Nous avons premièrement établi que ERK3 est exprimée chez les thymocytes. Ensuite, nous avons évalué le développement thymique chez la souris ERK3-déficiente et nous avons observé une diminution significative de la cellularité aux étapes DN1, DP et SP CD4+ du développement des LT. La création de chimères hématopoïétiques ERK3-déficientes nous a permis de démontrer que la diminution du nombre de cellules observée aux étapes DN1 et DP est autonome aux thymocytes alors que le phénotype observé à l’étape SP CD4+ est dépendant de l’abolition simultanée de ERK3 dans l’épithélium thymique et dans les thymocytes. Une étude plus approfondie de l’étape DP nous a permis de démontrer qu’en absence de ERK3, les cellules DP meurent plus abondamment et accumulent des cassures doubles brins (DSB) dans leur ADN. De plus, nous avons démontré que ces cassures dans l’ADN sont réalisées par les enzymes RAG et qu’en absence de ces dernières, la cellularité thymique est presque rétablie chez la souris ERK3-déficiente. Ces résultats suggèrent que ERK3 est impliquée dans un mécanisme essentiel à la régulation des DSB pendant le réarrangement V(D)J de la chaîne  du récepteur des cellules T (RCT). Dans le deuxième article présenté dans cette thèse, nous avons montré que ERK3 est exprimé chez les LT périphériques, mais seulement suite à leur activation via le RCT. Une fois activés in vitro les LT ERK3-déficients présentent une diminution marquée de leur prolifération et dans la production de cytokines. De plus, les LT ERK3-déficients survivent de façon équivalente aux LT normaux, mais étonnamment, ils expriment des niveaux plus faibles de la molécule anti-apoptotique Bcl-2. Ces résultats suggèrent que la prolifération réduite des LT ERK3-déficients est la conséquence d’une altération majeure de leur activation. Ainsi, nos résultats établissent que ERK3 est une MAPK qui joue des rôles essentiels et uniques dans le développement thymique et dans l’activation des lymphocytes T périphériques. Grâce à ces travaux, nous attribuons pour la toute première fois une fonction in vivo pour ERK3 au cours de deux différentes étapes de la vie d’un LT. / Classical MAP kinases (MAPK) play essential roles during T cell development and activation. ERK3 is a member of the MAPK family for which no physiological function has been described yet. Also, ERK3 is an atypical MAPK since its structure and mode of regulation are different from the conventional MAPK. Even today, the events leading to ERK3 activation and its substrates or partners are still largely unknown. We have studied in this thesis the role of ERK3 during T cell development and activation by using a mouse model in which ERK3 is not expressed. First, we have established that ERK3 is expressed in thymocytes. Next, we have evaluated thymic development in ERK3-deficient mice and we have observed a significant decrease in cell number at DN1, DP and CD4SP stages of T cell development. ERK3-deficient hematopoietic chimeras revealed that the DN1 and DP phenotype are T-cell autonomous, while abrogation of CD4SP development requires ERK3-deficiency in both thymocytes and thymic epithelium. By investigating further the DP stage, we have shown that ERK3-deficient DP thymocytes are more prone to apoptosis and also accumulate DNA double-strand breaks (DSBs). Moreover, we have shown that the increase DSBs are the direct consequence of RAG activity and that abolition of RAG almost restored thymic cellularity in ERK3-deficient mice. These results suggest that ERK3 is involved in an essential mechanism of DBSs regulation during TCR recombination. In the second article presented in this thesis, we have shown that ERK3 is expressed in peripheral T cell, but only when their TCR is activated. Also, ERK3-deficient T cells presented a strong reduction in proliferation and cytokine secretion following in vitro stimulation. Moreover, activated T cells lacking ERK3 are not more prone to death and surprisingly, they are unable to up-regulate the expression of the anti-apoptotic molecule Bcl-2 following TCR stimulation. These results suggest that the reduced proliferation of ERK3-deficient T cells is a consequence of their defective activation. Collectively, our results unveil essential and unsuspected roles for ERK3 in T cell development and activation. With this study, we establish for the first time an in vivo function for the atypical MAPK ERK3 in two different stages during T cell life.

Diss. (sammanfattning) Stockholm : Karolinska institutet, 2007. / Härtill 5 uppsatser.

Made available in DSpace on 2015-07-22T20:50:16Z (GMT). No. of bitstreams: 0 Previous issue date: 2009-04-29. Added 1 bitstream(s) on 2015-08-11T03:25:40Z : No. of bitstreams: 1 Publico-062a.pdf: 992519 bytes, checksum: 41750ff4e6e38ad6517d6c7541049844 (MD5). Added 1 bitstream(s) on 2015-08-11T03:25:40Z : No. of bitstreams: 2 Publico-062a.pdf: 992519 bytes, checksum: 41750ff4e6e38ad6517d6c7541049844 (MD5) Publico-062b.pdf: 1669897 bytes, checksum: 5ca81b8b1f9a75f45902de9ce4bc36f7 (MD5) / Coordenação de Aperfeiçoamento de Pessoal de Nível Superior (CAPES) / Fundação de Amparo à Pesquisa do Estado de São Paulo (FAPESP) / O GB vírus C (GBV-C) está constituído por uma fita única de RNA de polaridade positiva e pertence à família Flaviviridae. Possui uma seqüência e organização genómica parecida ao vírus da hepatite C, (HCV). A infecção pelo GBV-C não foi associada a nenhuma patologia, embora, na co infecção com o HIV, tenha sido associada a uma sobrevida maior e retardo no desenvolvimento da imunodeficiência. O efeito benéfico do GBV-C parece ser mediado por alterações na resposta imune celular; contudo, os possíveis mecanismos para explicar esse efeito ainda não foram esclarecidos. Neste trabalho investigamos a freqüência e características genotípicas assim como o impacto da infecção pelo GBV-C nos indivíduos infectados pelo HIV-1. No primeiro manuscrito examinamos os conhecimentos descritos na literatura referentes à coinfecção e propusemos algumas hipóteses para explicar esses efeitos. Posteriormente, descrevemos a taxa de infecção, a prevalência, incidência e características genotípicas do GBV-C nesta população. Assim, uma considerável freqüência de infecção pelo GBV-C foi observada e a análise filogenética dos isolados de GBV-C mostraram ser do genótipo 1 e 2. Foi observada também uma correlação inversa entre a carga viral do GBV-C e a carga viral do HIV na inclusão e um ano depois, assim como uma correlação positiva, mas não significativa, entre a carga viral do GBV-C e a contagem de linfócitos T CD4+. Finalmente, avaliamos o efeito da viremia pelo GBV-C na ativação celular em recém infectados pelo HIV-1. Os pacientes foram agrupados em GBV-C viremicos e não virémicos e foram avaliados para a contagem de linfócitos T, marcadores de ativação celular e carga viral do GBV-C e HIV-1. Foram realizadas análises de univariada e multivariada para identificar variáveis associadas com ativação celular. Demonstramos que a viremia pelo GBV-C foi correlacionada com uma diminuição da ativação celular nos indivíduos HIV positivos e este efeito mostrou se independente da carga viral do HIV. Assim, esta associação entre a replicação do GBV-C e menor ativação celular pode explicar, pelo menos em parte, a proteção conferida pelo GBV-C na progressão da doença nos indivíduos infectados pelo HIV-1. / GB virus C (GBV-C) is a single stranded positive sense RNA virus, which is a member of the Flaviviridae. It has a close sequence homology and genomic organization to hepatitis C virus (HCV). No disease has been associated with GBV-C infection but coinfection with human immunodeficiency virus (HIV) leads to improved morbidity and mortality for the HIV infected subjects. The mechanism of the beneficial effect of GBV-C appears to be mediated by alterations in the cellular immune response. In this study we investigated the frequency and genotyping characteristics as well as the impact of the GBV-C infection among recently HIV-1 infected individuals. In the first manuscript we examined the current knowledge concerning this co-infection and developed hypotheses to explain its effects. Subsequently, we described the rate of infection, the prevalence, incidence and genotypic GBV-C characteristics in this population. In that regard, a considerable frequency of GBV-C infection was observed and the phylogenetic analysis of the GBVC isolates revealed the predominance of genotypes 1 and 2. Also, it was observed an inverse correlation between GBV-C load and HIV-1 load at the enrollment and after one year of follow-up, and a positive, but not statistically significant, correlation between GBV-C load and CD4+ T lymphocyte counts. Finally, we have investigated the effect of GBV-C viremia on T cell activation in early HIV-1-infection. The volunteers were enrolled into two groups: GBV-C viremic and non viremic, all co-infected with HIV-1. They were evaluated for T cell counts, cellular activation markers, GBV-C RNA detection, and HIV-1 viral load. Non-parametric univariate and multivariate analyses were carried out to identify the variables associated with cellular activation. We demonstrated that the GBV-C viremia is correlated with a lower T cell activation in HIV-1-infected individuals and this effect was independent of HIV-1 viral load. The association between GBV-C replication and lower T-cell activation may explain, at least in part, the protection conferred by this virus against disease progression to immunodeficiency in HIV-1-infected patients. / TEDE / BV UNIFESP: Teses e dissertações

O escorpião Tityus serrulatus é considerado uma das espécies mais perigosas para os seres humanos no Brasil, e sua peçonha induz resposta inflamatória local e sistêmica. Neste projeto, tivemos como objetivo estudar a produção de mediadores inflamatórios, as vias de ativação celular e os receptores da imunidade inata responsáveis pelo reconhecimento da peçonha do escorpião T. serrulatus (TsV), bem como de suas toxinas. Nós demonstramos que TsV, e suas toxinas Ts1 e Ts6 induzem a produção de NO, IL-6 e TNF-? por células J774.1, as quais podem ser potencializadas pela presença de LPS. No entanto, Ts2 apresenta atividade anti-inflamatória por induzir produção de IL-10 e inibe a liberação de NO, IL-6 e TNF-?, induzida pelo LPS. Mostramos ainda que Ts2 ou Ts6 isoladas do TsV, além das citocinas, induzem a produção dos mediadores lipídicos (LTB4 e PGE2), e estes contribuem para o recrutamento de leucócitos para a cavidade peritoneal. Em conjunto, os nossos dados demonstraram que Ts2 e Ts6 induzem inflamação por mecanismos dependentes da produção de citocinas e mediadores lipídicos, e que Ts2 pode desempenhar papel regulador da resposta. No entanto, os mecanismos responsáveis pelo reconhecimento da peçonha e indução da liberação de mediadores inflamatórios por células de mamíferos, são desconhecidos. Assim, dando continuidade aos nossos estudos, demonstramos que os receptores TLR2, TLR4 e CD14 reconhecem TsV, e medeiam a produção de citocinas e mediadores lipídicos. Além disso, nós demonstramos que TsV ativa NF-?B dependente de MyD88, e o fator c-Jun, independente de MyD88. Semelhante ao TsV, a sua toxina majoritária, Ts1, induz a fosforilação de NF-?B dependente de MyD88, via reconhecimento por TLR2 e TLR4, enquanto a ativação c-Jun é via TLR4, mas independente de MyD88. Dentro deste contexto, nós propusemos o termo Padrões Moleculares Associados à Venenos (VAMP) para se referir às moléculas que são introduzidas no hospedeiro por picadas e são reconhecidas por receptores de reconhecimento padrão (PRRs), resultando em inflamação. Demonstramos ainda, a formação de corpúsculos lipídios (CLs) e a geração de eicosanóides, após o reconhecimento do TsV por TLR2 e TLR4. Nossos dados mostraram que a formação de eicosanóides se correlaciona com a formação dos CLs, que por sua vez são dependentes de TLR2 e TLR4, e da ativação de PPAR?, sugerindo que este receptor nuclear pode modular a produção de citocinas pró-inflamatórias. Assim, concluímos que PPAR? pode ser um candidato-alvo atrativo para novas estratégias terapêuticas para prevenção dos efeitos deletérios resultantes da intensa liberação sistêmica de mediadores inflamatórios após envenenamento. / Tityus serrulatus is the scorpion considered one of the most dangerous species to humans in Brazil, which venom induces local and systemic inflammatory response. In this project, we aimed to study In this project, we aimed to study the inflammatory mediators production, cell activation and receptors of innate immunity responsible for recognition of the venom of the scorpion T. serrulatus (TsV) as well as their toxins. We have demonstrated that TsV and their toxins Ts1 and Ts6 induce NO, IL-6 and TNF-? production in J774.1 cells, which may be potentiated by presence of LPS. However, Ts2 exhibits anti-inflammatory activity due induction of IL-10 production and inhibits the release of NO, IL- 6 and TNF-? induced by LPS. We also show that Ts2 or Ts6 isolated of TsV, besides of the cytokines, induce the production of lipid mediators (LTB4 and PGE2), and these mediators contribute to leukocytes recruitment into the peritoneal cavity. Taken together, our data demonstrated that Ts2 and Ts6 induce inflammation by mechanisms dependent on the production of cytokines and lipid mediators, and that Ts2 may play regulatory role on the cell response. Furthermore, continuing our studies, we demonstrated that TLR2, TLR4 and CD14 receptors recognize TsV, mediating cytokines and lipid mediators production. We also showed TsV MyD88- dependent activation of NF-?B, and a MyD88-independent activation of the factor c-Jun. Similar to TsV, the majority toxin Ts1 induces MyD88-dependent phosphorylation of NF-kB via TLR2 and TLR4 recognition, while the c-Jun activation is through TLR4 recognition, but independent of MyD88. Within this context, we propose the term Venom-Associated Molecular Pattern (VAMP), to refer molecules that are introduced into the host by st ings and recognized by PRRs, resulting in inflammation. Finally, we investigated the formation of lipid bodies (LBs) and generation of eicosanoids, through TsV recognition by TLR2 and TLR4. Our data showed that eicosanoid production correlates with the LBs formation, which are dependent on TLR2, TLR4 and PPAR? activation, suggesting that this nuclear receptor can modulate cytokines inflammatory. Thus, we suggest that PPAR? may be an attractive candidate target for novel therapeutic strategies to prevent the deleterious effects of intense systemic release of inflammatory mediators after envenomation.