Neste estudo são comparados os níveis de citocinas plasmáticas circulantes e as populações periféricas de células Treg CD4+CD25+, com base na expressão de FOXP3 e CTLA-4, e de células dendríticas (DCs) em indivíduos infectados por P. falciparum, P.vivax ou co-infectados por ambas as espécies e em controles saudáveis, porém expostos à malária, provenientes de uma área de transmissão instável na Amazônia brasileira. Amostras sangüineas de 76 pacientes infectados e de 18 controles expostos foram coletadas e processadas para a obtenção de células mononucleares. As populações celulares foram avaliadas por citometria de fluxo e os níveis de citocinas circulantes, pela técnica de ELISA de captura. A infecção aguda induziu aumento no percentual de células CD4+CD25+FOXP3+CTLA-4+ (p=0,0029; teste de Kruskal-Wallis) e redução no número absoluto de DCs (p=0,0008; teste de Kruskal-Wallis); mas esses efeitos ocorreram independente da espécie do parasito infectante. Entre os pacientes com malária vivax, 35-40% apresentaram baixa proporção de DCs que expressam a molécula co-estimulatória CD86. A única variável associada à baixa proporção de DCs CD86+ foi a proporção de células CD4+CD25+FOXP3+ que expressam CTLA-4. Em relação aos níveis de citocinas circulantes observou-se aumento nos níveis de IFN-<font face=\"Symbol\">&#947 na infecção por P. falciparum (p=0,0050; teste de Kruskal-Wallis). Apesar da concentração de IL-10 estar elevada em todos os indivíduos infectados em relação aos controles expostos (p<0,0001; teste de Kruskal-Wallis) esses níveis foram bem mais expressivos em indivíduos com malária vivax. Plasmodium falciparum e P. vivax parecem estimular diferentes padrões de resposta imune no hospedeiro, mesmo quando a comparação envolve somente indivíduos com malária não-complicada expostos a níveis semelhantes de transmissão de malária. / This study compares levels of circulating cytokines and peripheral-blood populations of CD4+CD25+ Treg cells, based on the expression of FOXP3 and CTLA-4, and dendritic cells (DCs) in individuals infected with P. falciparum, P. vivax or co-infected with both species and in healthy controls living in an area of unstable transmission of malaria in the Brazilian Amazon. Blood samples from 76 malaria patients and 18 malaria-exposed but non-infected controls were collected and processed to obtain mononuclear cells. Cell populations were characterized by flow cytometry and levels of circulating cytokines were measured by capture ELISA. Acute infection induced an increase in the proportion of CD4+CD25+FOXP3+CTLA-4+ cells (p= 0.0029, Kruskal-Wallis) and a decrease in the absolute number of DCs (p= 0.0008, Kruskal-Wallis), being both effects independent of the infecting parasite species. 35-40% of the P. vivax-infected subjects (but none in the other groups of subjects) had few circulating DCs expressing the co-stimulatory molecule CD86, a putative marker of DC activation. The only variable associated with a low proportion of CD86+ DCs was the proportion of CD4+CD25+FOXP3+ expressing CTLA-4. Analysis of circulating cytokine levels revealed increased levels of IFN- <font face=\"Symbol\">&#947 in P. falciparum infection (p= 0.0050, Kruskal-Wallis); although IL-10 levels were high in all infected individuals, compared with exposed controls (p<0.0001, Kruskal-Wallis), the increase was much more pronounced in vivax malaria. Plasmodium falciparum and P. vivax</i. appear to stimulate different patterns of immune response in humans, even when comparisons are limited to individuals with uncomplicated malaria exposed to similar levels of malaria transmission.

INTRODUÇÃO: A imunoterapia baseada em células dendríticas derivadas de monócitos (MoDCs) constitui uma estratégia promissora para o tratamento de indivíduos infectados pelo HIV. Devido à sua notória plasticidade, populações heterogêneas de MoDCs podem ser obtidas in vitro, dependendo das condições da cultura. Consequentemente, a capacidade dessas células em secretar citocinas e expressar moléculas que participam do processo de apresentação antigênica (MHC, moléculas de adesão e coestimuladoras) é variável, podendo interferir no perfil e eficácia da resposta imune induzida pela terapia. Em nosso laboratório foi desenvolvido um protocolo clínico de vacinação terapêutica baseada em MoDCs e HIV autólogo inativado para o tratamento de indivíduos cronicamente infectados pelo HIV, não expostos à terapia antirretroviral. Deste modo tornou-se oportuna uma investigação in vitro mais aprofundada sobre a produção viral e as características das MoDCs utilizadas como produto vacinal. OBJETIVOS: Caracterizar o produto vacinal constituído por vírus autólogo e MoDCs de indivíduos infectados pelo HIV utilizados em imunoterapia, com relação a aspectos fenotípicos e funcionais. MÉTODOS: Foram incluídos no estudo 17 indivíduos cronicamente infectados pelo HIV, participantes de um estudo clínico de fase I/II de imunoterapia com MoDCs. Células mononucleares do sangue periférico (PBMCs) foram obtidas a partir de leucaférese e parte do material foi utilizada para isolamento e expansão de HIV em sistema de cultura autólogo ou alogênico. Outra parte das PBMCs foi utilizada como fonte de monócitos para diferenciação em MoDCs imaturas que foram pulsadas ou não com o HIV quimicamente inativado pelo aldrithiol-2 (HIV-AT-2), denominadas respectivamente MoDCs HIV-AT-2 e MoDCs maduras, e posteriormente ativadas com citocinas pró-inflamatórias. MoDCs foram avaliadas fenotípica e funcionalmente quanto à expressão de moléculas de superfície, capacidade fagocítica, potencial migratório, produção de citocinas e habilidade em gerar resposta celular in vitro, avaliada por meio da capacidade em induzir proliferação, produção de citocinas e atividade citotóxica em linfócitos T autólogos. RESULTADOS: O rendimento de partículas virais foi mais elevado quando a expansão do HIV foi realizada em sistema alogênico em comparação ao sistema autólogo. Após estímulo para maturação, tanto MoDCs maduras quanto MoDCs HIV-AT-2 apresentaram aumento na expressão de moléculas de coestimulação, ativação e migração, comparado às MoDCs imaturas. Com relação à caracterização funcional, observamos que MoDCs foram capazes de fagocitar partículas de dextran-FITC, exibiram baixo potencial migratório e baixa produção de citocina polarizante para Th1. Ainda, observamos reduzida atividade citotóxica induzida tanto por MoDCs HIV-AT-2 quanto por MoDCs maduras. Por outro lado, MoDCs HIV-AT-2 promoveram proliferação de linfócitos T autólogos e maior polifuncionalidade em células TCD4+ e TCD8+ em comparação às MoDCs maduras. CONCLUSÃO: A produção de vírus autólogo através de sistema alogênico resulta em maior rendimento viral e potencial imunogênico. O produto vacinal composto por MoDCs HIV-AT-2 é capaz de induzir resposta polifuncional antígeno especifica in vitro / INTRODUCTION: Immunotherapy based on monocyte-derived dendritic cells (MDDCs) is a promising strategy for the treatment of HIV-infected individuals. Due their plasticity, using different combinations of cytokines cocktail in vitro it is possible to obtain a heterogeneous MDDCs population. Consequently the capacity of these cells to secrete cytokines and express molecules that participate in antigen presentation varies (MHC, adhesion and costimulatory molecules) and can interfere in the profile and efficacy of the immune response induced by this therapy. A clinical trial was conducted in our laboratory to evaluate a immunotherapy based on dendritic cells sensitized with autologous inactivated HIV for the treatment of antiretroviral naive chronically HIV-infected individuals. Therefore, it was a good opportunity to study deeply the virus production and expansion in vitro and to characterize MDDCs used as a vaccine. OBJECTIVE. To characterize MDDCs in context of their phenotype and function as well as investigating viral production and expansion in autologous and allogenic systems. METHODS: 17 patients underwent apheresis before vaccination and their peripheral blood mononuclear cells (PBMCs) were used for autologous virus production and expansion of the virus was carried out in both autologous and allogenic systems. Monocytes were differentiated into immature MDDCs that were pulsed/or not with autologous chemically (aldrithiol-2) inactivated HIV particles (HIV-AT-2). These pulsed (HIV-AT-2 MDDCs) and non-pulsed (mature MDDCs) cells were then activated by proinflammatory cytokines. Phenotypic (cell surface marker) and functional analysis (phagocytosis, transmigration and cytokines production) of MDDCs and their priming and stimulation of lymphocyte (proliferation, polyfunctionality and cytotoxicity) was performed using flow cytometry. RESULTS. Viral yield was higher when expanded in allogenic compared to autologous system. After stimulation with proinflammatory cytokines, both HIV-AT-2 MDDCs and mature MDDCs presented increased costimulation expression, activation and migratory molecules compared to immature MDDCs. Regarding to functional characterization, we observed that MDDCs were able to phagocytize FITC-Dextran and exhibitted a low migratory potential and low production of Th1 polarizing response cytokines. Moreover we observed reduced cytotoxic activity induced by HIV-AT-2 MDDCs and mature MDDCs. On the other hand we also observed that HIV-AT-2 MDDCs were capable of inducing proliferation and polyfunctionality of autologous CD4+ and CD8+ T-lymphocytes compared to mature MDDCs. CONCLUSION. Allogenic system was found to be more efficient in increased viral yield in relation to autologous system. Besides, virus expanded in allogenic system showed a more immunogenic profile. Vaccine product (HIV-AT-2 MDDCs) was able to induce antigen specific polyfunctional response

Os dermatófitos são um grupo de fungos que têm a capacidade de invadir o tecido queratinizado (pele, pêlos e unhas) de seres humanos e animais para produzir uma infecção denominada de dermatofitose. O Trichophyton rubrum é o principal patógeno causador de dermatofitose. As lesões causadas por estas espécies são crônicas e de carater pouco inflamatória. A doença apresenta evolução lenta e pacientes cronicamente infectados não respondem bem a terapia antifúngica. Assim como na maioria dos patógenos, o sistema imune inato é determinante na resposta antifúngica. Neutrófilos, macrófagos e células dendríticas constituem as células efetoras do sistema imune. A resposta imune aos dermatófitos ainda não está bem elucidada. Atualmente é aceito que a resposta imune mediada por células é responsável pelo controle da infecção. Poucos estudos têm focado a resposta imune inata a esses fungos. Assim, fomos estudar a interação de células dendríticas de pacientes com dermatofitose com conídios de T. rubrum. Nossos resultados mostraram que células dendríticas derivadas de monócitos (CDDM) foram capazes de fagocitar conídios de T. rubrum e ainda verificamos que estas células permaneceram viáveis após fagocitose. Quando analisamos a viabilidade dos conídios de T. rubrum, após 24 e 48 horas de interação com CDDM, verificamos que após 48horas houve um aumento no número de conídios viáveis quando estes foram fagocitados por células de pacientes, mostrando que CDDM de paciente não conseguem matar os conídios após fagocitose. Avaliamos a liberação de óxido nítrico por CDDM e a análise dos resultados mostrou que não houve diferença significativa na liberação de NO pela CDDM na presença de conídio de T. rubrum. Analisamos a expressão de moléculas co-estimulatórias como CD80, CD86, CD83, CD40 e HLA-DR em pacientes com dermatofitose e em indivíduos controle, observamos que não houve diferença na expressão dessas moléculas na presença de conídios de T. rubrum quando comparadas com culturas de CDDM sem conídios. Entretanto, houve uma diminuição do número de células de pacientes que expressam estas moléculas na presença de conídio de T. rubrum. Foi detectado um aumento significativo na secreção de TNF-&#945; e de IL-12 pelas CDDM de pacientes quando em contato com conídio de T. rubrum. Avaliamos a capacidade das CDDM de indivíduos controle e pacientes pulsadas com concentrações crescentes de tricofitina em ativar linfócitos T CD4 e também verificamos o perfil de citocinas secretadas pelos linfócitos T CD4 após proliferação. Os resultados demonstraram que as CDDM foram capazes de estimular a proliferação de linfócitos somente em pacientes com dermatofitose. Foi detectado um aumento significativo na secreção de IL-4 pelos LT CD4 de indivíduos controle. Nossos resultados sugerem uma diferença no perfil de secreção de citocinas em indivíduos controle e pacientes. Indivíduos controle não produzem IL-12 e estimulam preferencialmente linfócitos T CD4 secretores de IL-4. Por outro lado, células dendríticas de pacientes produzem IL-12 e induzem a ativação de linfócitos T produtores de IL-4 e IL-10 / The dermatophytes are a group of fungi that have the capacity to invade the keratinized tissue (skin, hair and nails) of humans and animals to produce an infection called dermatophytosis. The Trichophyton rubrum is the main causative pathogen of dermatophytosis. Injuries caused by these species are chronic inflammatory and little character. The disease shows slow evolution and chronically infected patients do not respond well to antifungal therapy. Like most pathogens, the innate immune system is crucial in the antifungal response. Neutrophils, macrophages and dendritic cells are the effector cells of the immune system. The immune response to dermatophytes is not yet well elucidated. Currently it is accepted that the immune response mediated by cells is responsible for controlling the infection. Few studies have focused on the innate immune response to these fungi. Thus, we study the interaction of dendritic cells from patients with dermatophytosis with conidia of T. rubrum. Our results showed that dendritic cells derived from monocytes (CDDM) were capable of phagocytosed conidia of T. rubrum and found that these cells remained viable after phagocytosis. When we analyze the viability of conidia of T. rubrum after 24 and 48 hours of interaction with CDDM shows that after 48hours an increase in the number of viable conidia when they were phagocytized by cells of patients, showing that CDDM not kill the patient after the conidia phagocytosis. Evaluated the release of nitric oxide by CDDM and analysis of results showed that there was no significant difference in the release of NO by CDDM in the presence of conidia of T. rubrum. We analyzed the expression of co-stimulatory molecules such as CD80, CD86, CD83, CD40 and HLA-DR in patients with dermatophytosis and in control subjects, we observed that there was no difference in expression of these molecules in the presence of conidia of T. rubrum compared with cultures of CDDM without conidia . However, there was a decrease in the number of cells of patients who express these molecules in the presence of conidia of T. rubrum. It was observed a significant increase in the secretion of TNF-&#945; and IL-12 by CDDM of patients when in contact with conidia of T. rubrum. Evaluate the capacity of individuals to control and CDDM patients pulsed with increasing concentrations of trichophytin to activate CD4 T lymphocytes and also see the profile of cytokines secreted by CD4 + T lymphocytes after proliferation. The results showed that the CDDM were able to stimulate the proliferation of lymphocytes only in patients with dermatophytosis. We observed a significant increase in the secretion of IL-4 by CD4 L T to control individuals. Our results suggest a difference in the profile of secretion of cytokines in control subjects and patients. Control subjects did not produce IL-12 and preferentially stimulate CD4 T lymphocytes secreting IL-4. Furthermore, dendritic cells of patients produce IL-12 and induce the activation of T lymphocytes producing IL-4 and IL-10

Streptococcus pneumoniae ist ein gram-positives Bakterium und ein Krankheitserreger des Menschen. Ein Charakteristikum des Bakteriums ist, dass es Cholin-Reste in seine dicke Zellwand einbaut. Das Ziel meiner Doktorarbeit war herauszufinden, inwiefern diese Cholin-Reste zur Virulenz des Bakteriums während experimenteller Sepsis und Meningitis beitragen. Dabei konnte ich feststellen, das cholinierte Wildtyp-Bakterien hoch virulent waren, ungestört im Wirt wachsen konnten und letztendlich zu einer massiven Überaktivierung des Wirts-Immunsystems (gemessen anhand der Zytokine IL-1beta, IL-6, IL-12, TNFalpha) sowie zum Tode der Versuchtiere führten. Im Gegensatz dazu waren cholin-freie Bakterien nicht in der Lage eine permanente Infektion im Wirt zu etablieren. So wuchsen sie nur anfangs und wurden vom Wirts-Immunsystem kontrolliert und beseitigt, sodass alle Tiere überlebten. Die Injektion von cholin-freien und cholinierten, hoch aufgereinigten Zellwänden, führte zu der Schlussfolgerung, dass Cholin in der Zellwand ein Immunevasionsmechanismus der Bakterien sein muss. Ausserdem waren cholin-freie Bakterien in der Lage einen protektiven, serotyp-spezifischen Immunschutz im Wirt zu induzieren. / Streptococcus pneumonia is a major human pathogen. Since it is a gram positive bacterium it is characterized by the production of a thick cell wall. Being auxotrophic for choline, the pneumococcus attaches this aminoalcohol to its teichoic acids thus decorating its surface with choline-residues. The aim of this work was to investigate the role that these choline residues play in the virulence of the bacterium. By using an isogenic choline-containing and choline-free pair of S. pneumoniae I was able to demonstrate that surface bound choline is essential for the virulence of the bacterium in animal models of experimental sepsis and meningitis. In either model choline-containing bacteria were able to persistently grow within the host system, continuously stimulate the production of proinflammatory cytokines (IL-1beta, IL-6, IL-12, TNFalpha) and eventually led to the death of all infected animals within 24h. In contrast, choline-free bacteria showed only transient growth within the host and induced only moderate and limited expression of cytokines. Consequently, the bacterium was virtually avirulent and all animals survived. Intracisternal application of highly purified cholinated and choline-free cell wall preparations, induced a comparable activation of the immune system. These findings led to the conclusion that choline residues contribute to an immunevasion strategy that allows the bacteria to grow despite an ongoing immune response. Although being avirulent choline-free bacteria were able to induce serotype specific immunity in the animals.

Bei der Kreuzpräsentation durch dendritische Zellen (DC) werden extrazelluläre Antigene in den MHC I-Präsentationsweg eingeschleust und CD8+ T-Zellen präsentiert. Bisher werden die kreuzpräsentierenden DC der Maus durch die Expression der Moleküle CD8 in der Milz und CD103 in der Peripherie abgegrenzt; der kreuzpräsentierende Subtyp primärer humaner DC war vor Beginn dieser Arbeit nicht bekannt. Innerhalb dieser Arbeit konnte erstmals eine durchflusszytometrische Färbung von XCR1 auf der Oberfläche von Zellen etabliert werden, wodurch demonstriert wurde, dass XCR1 auf 83 % der CD8+ und 4 % der CD8-CD4- DC der Milz exprimiert wird. Der Phänotyp der XCR1+ DC der Milz unterschied sich deutlich von dem der XCR1- DC. In Milzen von Batf3- und Irf 8-defizienten Mäusen konnten keine XCR1+ DC detektiert werden. Zudem wurde nach Applikation des Wachstumsfaktors Flt3 Ligand in C57BL/6 Mäusen der Anteil der XCR1+ DC signifikant erhöht. Diese Ergebnisse verdeutlichen, dass die Entwicklung der XCR1+ DC von diesen Faktoren abhängig ist. Funktionell waren die XCR1+ DC wesentlich effektiver in der Kreuzpräsentation von löslichen und zellassoziierten Antigenen. Damit kann XCR1 als Oberflächenmarker verwendet werden, um murine kreuzpräsentierende DC zu kennzeichnen. Um herauszufinden, ob XCR1+ DC auch im Menschen existieren, wurde die Expression von XCR1 auf Zellen des humanen peripheren Blutes anhand von qPCR und Durchflusszytometrie untersucht. Ausschließlich CD141+ DC exprimierten XCR1-mRNA und -Protein. Durch die Etablierung einer effektiven Sortierungsstrategie zur Isolierung aller DC-Subtypen konnte erstmals gezeigt werden, dass die CD141+ DC den einzigen effektiven kreuzpräsentierenden DC-Subtyp des Blutes darstellen. XCR1 ist somit auch im humanen System spezifisch auf kreuzpräsentierenden DC exprimiert. Zusammengefasst zeigen die Ergebnisse, dass es sich bei den XCR1+ DC der Maus und des Menschen um funktionelle Homologe handelt. / Cross-presentation by dendritic cells (DC) is a process, in which extracellular antigen is shunted into the MHC I presentation pathway and presented to CD8+ T cells. So far, murine cross-presenting DC were defined by the expression of the molecules CD8 in the spleen and CD103 in the periphery. However, cross-presenting DC have not been characterized in humans. In this work, a flow cytometric staining of XCR1 was established for the first time which allowed the detection of XCR1 on 83 % of CD8+ DC and 4 % of CD8-CD4- DC of the spleen. The phenotype of splenic XCR1+ DC differed markedly from XCR1- DC. Both, Batf3- and Irf 8-deficient mouse strains showed an absence of splenic XCR1+ DC. Furthermore, the frequency of XCR1+ DC was significantly increased in spleens of C57BL/6 mice treated with Flt3 ligand. These results demonstrate that the development of XCR1+ DC is dependent of these factors. To test the ability to cross-present antigen, all splenic conventional DC were sorted according to their expression of CD8 and XCR1. XCR1+ DC were most efficient in cross-presenting soluble and cell-associated antigen to CD8+ T cells. Therefore, XCR1 is the first surface marker that can be used to delineate murine cross-presenting DC and the development of this distinct DC population is strongly dependent on Batf3, IRF-8 and Flt3 ligand. To explore if XCR1+ DC also exist in men, cell populations of human peripheral blood were analysed for their XCR1-expression using qPCR and flow cytometric staining. Only CD141+ DC express XCR1 mRNA and protein. An efficient sorting strategy for the isolation of all DC subsets was established to compare their ability to cross-present soluble and cell-associated antigen. CD141+ DC were the only effective cross-presenting DC subtype. Therefore, XCR1 is also in the human a receptor expressed specifically on cross-presenting DC. In summary, the data show that the XCR1+ DC of mouse and men are functional homologues.

En transplantation d’organes, la réponse immunitaire de l’hôte contre son donneur reste une cause très importante de perte de greffons. Ainsi, un meilleur contrôle de la réponse par induction d’une tolérance spécifique reste une priorité en transplantation humaine. Dans ce projet,nous avons donc étudié l’homéostasie calcique au cours des phénomènes précoces de la maturation des cellules dendritiques (DC) humaines. Nous avons mis en évidence la présence d’une ECC (Entrée Capacitive de Calcium), dans la DC humaine, lors d’une stimulation par des agonistes des TLR (LPS ou Zymosan) ou des cytokines inflammatoires (TNF-α). De plus, cette ECC est gouvernée par le complexe ORAI-1 / STIM-1. En effet, l’absence d’expression de ces protéines induit une diminution de la maturation des DC humaines (phénotype, synthèse de cytokines). Par ailleurs, le Calcium, entré par l’ECC, va induire l’activation de canaux potassiques, sensibles à la concentration intracellulaire en Calcium : les canaux KCa3.1. Ces canaux, outre leur rôle dans la facilitation de l’ECC par hyperpolarisation de la membrane,contrôlent la migration des DC humaines. En effet, leur activation permet une sorte d’état de quiescence où les DC restent dans les tissus périphériques. L’ensemble de ces résultats obtenus,in vitro, suggèrent que ces canaux ioniques pourraient être une cible privilégiée pour le développement de nouvelles thérapies pour l’induction de tolérance immune en transplantation. / In organ transplantation, host immune response against donor still remains a major causeof graft loss. A better control of allogeneic response through the induction of specific tolerance isa major goal in human transplantation. In this work, we studied the calcium homeostasis during the earlier events of the human dendritic cells (DC) maturation. We showed the presence of a CCE(Capacitative Calcium Entry), in the human DC, during the stimulation by TLR agonists (LPS andZymosan) or inflammatory cytokines (TNF-α). Moreover, this CCE was managed by the ORAI-1/ STIM-1 complexe. Indeed, an inhibition of these proteins expression induced a decrease in theDC maturation (phebnotype, cytokines production). Then, the Calcium, issued to CCE, could activate potassium channels, sensitive to intracellular Calcium concentration: KCa3.1 channels.These channels, excepted their role in the facilitation of CCE by membrane hyperpolarisation,control the capacity of human DC migration. Their activation induced a kind of steady state where the DC staied in peripheral tissues. These results taken together suggest that the ion channels seemto be a good target for the development of new therapies to in order to promote allograft tolerance.

Notre système immunitaire a pour fonction de nous protéger des infections mais également des allergies, des cancers ou des maladies inflammatoires. Les cellules dendritiques (DC) sont au coeur de ce système en orientant les réponses immunitaires vers une voie effectrice ou tolérogène. A l’état immature, elles sont présentes, entre autres, aux interfaces avec le milieu extérieur. Elles permettent ainsi la reconnaissance des antigènes, notamment ceux présents dans le tube digestif, comme les aliments ou la flore commensale. Certaines bactéries dites probiotiques sécrètent des métabolites au cours de la fermentation définissant les produits de fermentation. Notre travail montre qu’un produit de fermentation de la souche bactérienne BbC50 (BbC50sn) activent les DC humaines grâce à sa fixation sur le récepteur TLR2 et modulent de nombreux récepteurs à leurs surfaces. Les DC ainsi maturées peuvent activer des lymphocytes T CD4+ et CD8+ et également induire des lymphocytes T régulateurs CD4+ et CD8+. / The immune system protects an organism from infections, allergy, cancer and inflammatory diseases. Dendritic cells (DC) present antigens to T cells and polarize lymphocytes toward either an effector or a tolerogenic response. In an immature state, DC are localized in mucosas at interfaces with the external environment, after receiving signals they acquire the ability to activate T cells. Thus, DC allow the recognition of antigens, present in the digestive tract, such as food or commensal flora. Some probiotic bacteria produce metabolites during fermentation defined as the fermentation products. Our work has shown that a fermentation product of the bacterial strain BbC50 (BbC50sn) activate human DC through TLR2 ligation on DC and modulate numerous receptors on their surfaces. DC activated with this fermentation product activate CD4+ and CD8+ T cells and also induce CD4+ and CD8+ regulatory T lymphocytes.

Le rôle de l'interleukine 2 (IL-2) dans les cellules dendritiques (DC) humaines n’est pas bien défini. Dans ce travail, nous avons exploré les effets de l'IL-2 sur des DC dérivées de monocytes. Nous démontrons une expression constitutive des chaines des β et γ du récepteur de l’IL-2 sur les DC ainsi qu’une une expression inductible de la chaîne α en réponse à des agents ‘maturants’ tels le LPS et le TNFα. L’IL-2 induit la phosphorylation du facteur de transcription STAT5 provoquant l’augmentation de la synthèse d'IFN-γ par les DC sans modifier leur phénotype ou leur survie. En revanche, nous n'avons pas mis en évidence d’activation des autres voies de signalisation du récepteur de l’IL-2. Nous avons également démontré que l'IL-2 augmente les capacités des DC à activer les lymphocytes T CD4+ allogéniques et les lymphocytes T CD8+ indépendamment des lymphocytes T auxilliaires. Nous n’avons pas pu mettre en évidence la sécrétion endogène d'IL-2 par les DC cependant les anticorps anti-CD25 diminuent les capacités allostimulatrices de DC en absence d’IL-2 exogène. Ainsi nos travaux indiquent que l’expression de CD25 par les DC matures est un événement clé menant la DC à un nouvel état d’activation. / Human dendritic cells (DC) express interleukin 2 (IL-2) receptor α-chain (CD25), but the role of IL-2 in DC is poorly understood. In this work, we explored the effects of IL-2 on monocyte-derived DC. First, we demonstrated the constitutional expression of β and γ chain of IL-2R on DC, while the α-chain was inducible by LPS and TNFα. Then we found that IL-2 does not affect DC phenotype and apoptosis but increases IFN-γ synthesis in DC through activation of transcription factor STAT5. Moreover we reported that IL-2 increases the ability of DC to activate allogeneic CD4+ T cells and helpless CD8+ T lymphocytes, most likely because of IL-2–triggered IFN-γ synthesis. We have not been able to demonstrate the endogenous secretion of IL-2 by DC, however anti-CD25 decreased allostimulatory capacity of DC in the absence of exogenous IL-2.In summary, we disclose that IL-2 induces DC functional maturation and activation. Interestingly, our study suggests a direct effect of anti-CD25 monoclonal antibodies on DC and that CD25 expression regulation on human DC could be used to control immune response in vivo.

Les cellules dendritiques (DCs) détectent les particules virales et présentent les antigènes viraux afin d’organiser la réponse immunitaire. La réplication virale dans les DCs induit une réponse immune cytosolique. Comment les DCs tolèrent les virus afin de maintenir leur intégrité fonctionnelle est inconnu. Les DCs sont organisées en sous-populations distinctes d’un point de vue ontogénique. Nous avons observé que le virus du VIH et de la grippe infectaient préférentiellement les DCs CD1c+ par rapport au DCs CD141+ et aux pDCs. La réplication de ces virus au sein des DCs CD1c+ est essentielle afin d’établir une activation efficace des lymphocytes T CD8+ et d’assurer une détection cytosolique. Les DCs CD141+ et les pDCs, quant à elles, répondent aux virus exogènes. L’étape de fusion virale virale est constitutivement réduite dans les DCs CD141+ et les pDCs en comparaison des DCs CD1c+. La petite GTPase RAB15 est exprimée sélectivement dans les DCs CD141+ et les pDCs et contribue à la résistance de ces deux sous-populations de DCs au VIH et à la grippe. La résistance sélective des sous-populations de DC à l’infection virale pourrait représenter un mécanisme de tolérance afin d’augmenter la réponse antivirale. / Dendritic cells (DCs) sense viral particles and present viral antigens to induce immune responses. Viruses also replicate in DCs, engaging cytosolic immune responses. How DCs tolerate viruses to ensure functional integrity is unknown. DCs are developmentally organized in distinct subsets. We find that HIV and influenza preferentially infect CD1c+ DCs over CD141+ DCs and pDCs. Replication in CD1c+ DCs was essential for efficient CD8+ T cell activation and cytosolic sensing, while CD141+ DCs and pDCs responded to exogenous virus. Viral fusion was constitutively reduced in CD141+ and pDCs compared to CD1c+ DCs. The small GTPase RAB15 expressed selectively in CD141+ and pDCs contributed to the resistance. Selective resistance of DC subset to viral infections may thus represent a tolerance mechanism to maximize antiviral responses.

Infektionen mit dem intestinalen Parasiten Giardia duodenalis, verursachen weltweit eine der häufigsten humanen Parasitosen. Bislang konnten keine eindeutigen Virulenz- oder Pathogenitätsmarker des Erregers beschrieben werden. Es wird allerdings vermutet, dass potentielle G. duodenalis Virulenzfaktoren Enzyme sind, die während des Kontaktes des Erregers mit den Dünndarmepithelzellen sezerniert werden. Eines dieser Enzyme ist die Arginin Deiminase (ADI), die Arginin zu Citrullin umwandelt. Ziel dieser Arbeit war es Merkmale zu identifizieren, die für die ADI als Virulenzfaktor sprechen. Dazu wurde das Enzym zunächst hinsichtlich seiner Bedeutung für die Wirt-Pathogen-Interaktion untersucht. Die mit rekombinanter, katalytisch aktiver ADI (Assemblage A) behandelten LPS-stimulierten humanen moDC zeigten eine Veränderung in ihrem Phänotyp als auch in ihrer Cytokinsekretion. Diese ließ sich auf die durch das Enzym hervorgerufene Arginindepletion und/oder auf die Bildung der Metabolite, Citrullin und NH4+, zurückführen. Weiterhin konnte gezeigt werden, dass Parasitenisolate verschiedener G. duodenalis Assemblage A-Subtypen, vermutlich durch die katalytische Aktivität der ADI, die Stickstoffmonoxid-Bildung einer intestinalen Epithelzelllinie inhibiert. Neben dem Einfluss auf die Wirtsimmunantwort wurde auch die Variabilität in der kodierenden Sequenz des Enzyms in verschiedenen Parasitenisolaten analysiert. Anschließend erfolgte die funktionelle Charakterisierung des nativen (verschiedene Assemblage A-Subtypen) als auch des rekombinant aufgereinigten Enzyms (Assemblage A, B und E). Dabei zeigten sich Unterschiede in der Substrataffinität der ADI für Arginin, sowohl zwischen unterschiedlichen Assemblage A-Subtypen als auch unterschiedlichen Assemblage-Klassen. Zusammenfassend wurde gezeigt, dass die G. duodenalis ADI immunmodulatorische Effekte hat und das vermutlich eine Korrelation zwischen der Variation in der Primärstruktur und der Funktion des Enzyms besteht. / Giardia duodenalis (G. duodenalis) is an intestinal protozoan parasite that causes giardiasis, one of the most prevalent parasitic diseases worldwide. So far, little is known concerning host-parasite interaction, in particular what determines the parasite’s pathogenicity. Several potential virulence factors, among them the arginine deiminase (ADI) that hydrolyzes arginine into citrulline and NH4+, are discussed. The ADI was identified to be released upon contact with intestinal epithelium by Giardia trophozoites and was recognized as an immunoreactive protein during acute human giardiasis. Aim of the study was to identify hints for G. duodenalis ADI to be a virulence factor. First, to analyze the enzyme’s impact on host-parasite interplay, its influence on human monocyte-derived dendritic cells (moDC) was investigated. Treatment of LPS-stimulated cells with recombinant ADI of assemblage A changed DC phenotype (CD83, CD86) and cytokine secretion (TNF-α, IL-10, IL-12p40). These immunomodulatory changes in DC response were due to arginine depletion and the formation of reaction products, in particular, ammonium ions. Furthermore, trophozoites of different assemblage subtypes were shown, probably due to consumption of arginine by ADI, to reduce nitric oxide formation by intestinal epithelial cells in vitro. Second, variation in the ADI coding sequence of different G. duodenalis isolates being collected in a Giardia biobank was analyzed by sequencing. Subsequently, functional genetics were performed with native ADI of different assemblage A subtypes expressed by these strains as well as with purified, recombinant ADI of assemblage A, B and E. It was recognized that enzymes of the same subtype as well as of different assemblages types had different substrate affinities for arginine. To sum up, this report identified G. duodenalis ADI to be immunomodulatory and gives first indications of a correlation between enzyme function and variation of the protein primary structure.