O veneno da subespécie de cascavel Crotalus durissus terrificus (C.d.terrificus) altera a coagulação, tem ação neurotóxica, miotóxica e efeito imunossupressivo. O veneno de C.d.terrificus e a sua fração majoritária, crotoxina (CTX), inibem a resposta celular e humoral, sendo esse efeito independente da indução de morte celular. O objetivo foi investigar os mecanismos envolvidos na imunossupressão exercida pela CTX. A CTX induz a produção de IL-10, TGF-<font face=\"Symbol\">&#946 e prostaglandina E2 (PGE2) pelas células dos camundongos que a receberam. Menor secreção de IFN-<font face=\"Symbol\">&#947 e IL-12 foi observada nas culturas de células de camundongos imunizados e que receberam CTX. A CTX aumenta a expressão da enzima indoleamina 2,3 dioxigenase e a geração de células T reguladoras em camundongos imunizados com OVA e que receberam CTX. A CTX foi capaz de inibir a expressão de CD40, CD80, CD86 e de MHC de classe II em células de camundongos imunizados e nas células dendríticas (DCs) purificadas destes animais. Em culturas de DCs, a CTX induz alta secreção de IL-10, TGF-<font face=\"Symbol\">&#946 e PGE2 e menor de IL-12. / The venom of the rattlesnake Crotalus durissus terrificus (C.d.terrificus) changes the coagulation system and presents neurotoxic, myotoxic and immunosuppressive effect. The venom and its main fraction, crotoxin (CTX) inhibited both the cellular and humoral response. This effect is not due to induction of cell death. The objective was to investigate the immunosuppressive mechanisms of CTX. CTX induces production of IL-10, TGF-<font face=\"Symbol\">&#946 and prostaglandin E2 (PGE2) by cells of mice that received it. Lower secretion of IFN-<font face=\"Symbol\">&#947 and IL-12 was observed in the cultured cells from mice immunized that received CTX. CTX promotes increased expression of the enzyme indoleamine 2,3 dioxygenase and generation of regulatory T cells in mice immunized with OVA and receiving the toxin. CTX was able to inhibit the expression of CD40, CD80, CD86 and MHC II in mice immunized and in dendritic cells (DCs) purified from these animals. In cultures of DCs, CTX increased secretion of IL-10, TGF-<font face=\"Symbol\">&#946 and PGE 2 and lower IL-12.

Paracoccidioidomicose (PCM), micose sistêmica prevalente na América Latina, é uma doença granulomatosa causada pelo fungo termodimórfico do gênero Paracoccidioides spp. O maior número de casos dessa doença tem sido reportado no Brasil, Colômbia, Venezuela e Argentina. A existência de extensas áreas endêmicas, alto potencial incapacitante devido a fibroses pulmonares, grande quantidade de mortes prematuras, faz com que a PCM seja considerada um grave problema de Saúde Pública. Apesar do tratamento medicamentoso ser relativamente eficiente, o tempo longo de uso das drogas e os efeitos colaterais provocados podem levar a diminuição da adesão ao tratamento diminuindo assim eficácia. O uso de vacinas terapêuticas pode ser uma importante ferramenta no controle da PCM. Uma das modalidades investigadas pelo nosso grupo é a utilização de células dendríticas (DCs) pulsadas com o peptídeo P10 como vacina terapêutica no controle da PCM. Resultados apresentados por nosso grupo, demonstraram que DCs diferenciadas a partir de células de medula óssea de camundongos saudáveis pulsadas com o peptídeo 10 (P10) podem ser utilizadas como adjuvante no tratamento da paracoccidioidomicose experimental em camundongos imunocompetentes e imunossuprimidos. Assim sendo, nossa proposta no presente trabalho foi avaliar se DCs diferenciadas de células de medula óssea (BMDCs) ou de monócitos circulantes (MoDCs) de camundongos (BALB/c) infectados apresentam modulação prévia devido à exposição a antígenos fúngicos, avaliamos in vitro a capacidade das células dendríticas diferenciadas a partir de monócitos circulantes de camundongos infectados com Pb18 estimular proliferação de linfócitos CD8+ e CD4+, analisamos a capacidade de células dendríticas derivadas de monócitos circulantes de camundongos infectados com Pb18 pulsadas com P10 em induzir uma resposta protetora. Nossos resultados obtidos são promissores e reforçam dados já publicados pelo grupo os quais demonstraram a eficiência do peptídeo P10 no tratamento da paracoccidioidomicose experimental; apontam que o P10 é capaz de modular ativamente células dendríticas diferenciadas de monócitos circulantes e diferenciadas de células de medula óssea; demonstram que BMDCs e MoDCs pulsadas ou não com P10 tem a capacidade de estimular proliferação de linfócitos T CD4+ e CD8+; confirmaram que BMDC e MoDCs pulsadas ou não com P10 diminuem a carga fúngica no tecido pulmonar, estimulando um padrão de citocinas misto, porém com predominância de citocinas pró-inflamatórias que incitam resposta imune celular do tipo Th1, que é protetora na PCM. Estes dados foram respaldados com a análise dos cortes histológicos onde os pulmões dos camundongos tratados com BMDCs ou MoDCs pulsadas com P10 apresentaram parênquima pulmonar mais conservado em comparação com os pulmões dos grupos que não receberam tratamento com BMDCs ou MoDCs, com diminuição significativa de células fúngicas viáveis / Paracoccidioidomycosis (PCM), a systemic mycosis prevalent in Latin America, is a granulomatous disease caused by the thermodymorphic fungus of the genus Paracoccidioides spp. The highest number of cases of this disease has been reported in Brazil, Colombia, Venezuela and Argentina. The existence of extensive endemic areas, a high incapacitating potential due to pulmonary fibroses, and a large number of premature deaths, makes the PCM considered a serious public health problem. Although the drug treatment is relatively efficient, the long time of use of the drugs and the side effects provoked can lead to the decrease of the adherence to the treatment thus diminishing their effectiveness. The use of therapeutic vaccines can be an important tool in the control of PCM. One of the modalities investigated by our group is the use of dendritic cells (DCs) pulsed with the peptide P10 as therapeutic vaccine in the control of PCM. Results presented by our group demonstrated that DCs differentiated from bone marrow cells of healthy mice pulsed with peptide 10 (P10) can be used as an adjuvant in the treatment of experimental paracoccidioidomycosis in immunocompetent and immunosuppressed mice. Thus, our proposal in the present study evaluated the capacity of differentiated DCs of bone marrow cells (BMDCs) or circulating monocytes (MoDCs) of infected mice (BALB/c) exhibited previous modulation due to exposure to fungal antigens, we evaluated in vitro the ability of differentiated dendritic cells from circulating monocytes from Pb18 infected mice to stimulate proliferation of CD8+ and CD4+ lymphocytes, we analyzed the ability of dendritic cells derived from circulating monocytes from mice infected with P. brasiliensis pulsed with P10 to induce a protective response. Our results are promising and reinforce data already published by the group which demonstrated the efficiency of the peptide P10 in the treatment of experimental paracoccidioidomycosis; suggest that P10 is capable of actively modulating differentiated dendritic cells from circulating and differentiated monocytes from bone marrow cells; demonstrate that BMDCs and MoDCs pulsed or not with P10 have the ability to stimulate proliferation of CD4+ and CD8+ T lymphocytes; confirmed that BMDC and MoDCs pulsed or not with P10 decrease the fungal load in lung tissue, stimulating a pattern of mixed cytokines but with a predominance of pro-inflammatory cytokines that stimulate Th1-type cellular immune response that is protective in PCM, these data were supported with analysis of the histological sections where the lungs of the mice treated with BMDCs or MoDCs pulsed or not with P10 showed more conserved pulmonary parenchyma compared to the lungs of the groups that did not receive treatment with BMDCs or MoDCs, with significant decrease of viable fungal cells

Nous avons découvert que les DC pathologiques (LCH DC) qui s’accumulent dans les granulomes de patients atteints d’histiocytose langerhansienne (LCH) produisent l’IL-17A (Coury et al, Nat Med 2008). In vitro, les LCH DC fusionnent en cellules géantes (MGC) sous l’influence de leur production autocrine d’IL-17A. In vivo, les granulomes de LCH DC et MGC détruisent les tissus. Pendant la thèse, nous avons étudié les transcriptomes des monocytes, DC, traitées ou non par l’IL-17A et des LCH DC. L’IL-17A induit BCL2A1, un membre de la famille Bcl-2 qui prolonge la survie des DC. Elle induit aussi les chémokines CCL20 et CCL2, qui regroupent les DC avant leur fusion. L’induction du récepteur nucléaire LXR-α, de protéines impliquées dans le métabolisme, le transport et le stockage des lipides témoignent d’un profond remodelage lipidique. Après la confirmation de ces régulations par PCR et western, les goutelettes lipidiques sont quantifiées à l’huile rouge, puis l’analyse lipidomique révèle l’augmentation de phospholipides, triglycérides, esters de cholestérol et cholestérol par l’IL-17A dans les DC qui sont aussi capables de capturer du palmitate extracellulaire fluorescent. Les transcriptomes des DC traitées à l’IL-17A et des LCH DC sont similaires. La simvastatine qui bloque la synthèse de cholestérol tue les LCH DC. Ce travail établit pour la première fois que l’IL-17A affecte profondément le métabolisme lipidique des DC, une activité qui pourrait avoir d’importantes applications dans les maladies chroniques inflammations associées à une dérégulation lipidique comme l’athérosclérose, l’obésité, la tuberculose et la LCH / We found that the pathological DCs (DC LCH), accumulating in the granulomas of patients affected with Langerhans cell histiocytosis (LCH), produce IL-17A (Coury et al, Nat Med 2008). In vitro, LCH DCs form multinucleated giant cells (MGC) by a fusion process, under the influence of their IL-17A autocrine production. In vivo, granulomas, which are mainly composed of LCH DCs and MGCs, destroy tissues of the patients. During the PhD, we studied the transcriptomes of monocytes, DCs, treated or not with IL-17A, and LCH DCs. IL-17A induceed BCL2A1, a member of the Bcl-2 family that prolonged DC survival. It also induced the CCL2 and CCL20 chemokines that clustered DC, a process required to license DC fusion. The inductions of LXR-α nuclear receptors, proteins involved in metabolism, transport and storage of lipids signed a deep lipid remodeling induced by IL-17A in DCs. We confirmed these regulations by PCR and western studies, then the lipid droplets were quantified after Oil Red-O staining. Further lipidomic analysis revealed an increase of phospholipids, triglycerides, cholesteryl esters and cholesterol by IL-17A in DCs, which are also able to capture extracellular fluorescent palmitate. Transcriptomes of DCs treated with IL-17A and LCH DCs were similar. Simvastatin, which inhibits the synthesis of cholesterol, killed LCH DC. For the first time, this work establishes that IL-17A profoundly affects the lipid metabolism of DCs, an activity that may have important applications in chronic inflammations associated with lipid deregulations such as atherosclerosis, obesity, tuberculosis and LCH

Une fois activées, les cellules dendritiques (DCs) migrent dans les organes lymphoïdes ou elles exercent leur rôle de cellules présentatrices d’antigène professionnelles. Elles sont capables d’activer et d’induire la différenciation des lymphocytes T naïfs en différentes sous-populations de lymphocytes T auxiliaires. L’ajustement de la réponse lymphocytaire T au type d’inflammation est assuré par les DCs à deux niveaux. Premièrement, grâce à leur plasticité, les DCs adaptent leur comportement en fonction de la combinaison de signaux issus du microenvironnement inflammatoire. Deuxièmement, il existe différentes sous-populations de cellules dendritiques ayant de différentes spécialisations fonctionnelles.Mon travail de thèse s’est concentré sur l’étude de la diversité des réponses des sous-populations de cellules dendritiques humaines suite à la stimulation par la cytokine lymphopoïetine stromale thymique (TSLP). Cette cytokine est secrétée par les cellules épithéliales et la peau au cours de l’inflammation. La TSLP active principalement les DCs myéloïdes, induisant celles-ci à secréter les chimiokines inflammatoires CCL17 et CCL22. Les DCs activées par la TSLP (TSLP-DCs) induisent une réponse inflammatoire de type Th2, et sont impliquées dans le développement de l’allergie. La comparaison systématique de la réponse à la TSLP par les sous-populations de DCs du sang, BDCA1+ et BDCA3+, nous a permis de montrer que ces deux sous-populations sont activées par la TSLP. Toutefois, nos résultats montrent que la TSLP, en synergie avec le TGF-β, induit la différenciation des DCs BDCA-1+ en cellules de Langerhans, mais pas celle des DCs BDCA-3+. De plus, la TSLP induit la migration cellulaire et la sécrétion de chimiokines seulement chez la sous-population de DCs BDCA-1+. Des analyses complémentaires des mécanismes impliqués dans la migration des DCs BDCA-1+ en réponse à la TSLP révèlent que, d’une part la TSLP est indispensable à l’induction de la migration et d’autre part, qu’un récepteur de chimiokines, sensible à la Toxine Pertussique serait impliqué. Au final, nos résultats révèlent (i) de nouvelles capacités des DCs en tant que cellules précurseurs, (ii) de différentes propriétés fonctionnelles des sous-populations de DCs en réponse à la stimulation par la TSLP, (iii) et démontrent la complexité des mécanismes impliqués dans la migration des DCs induite par la TSLP. / Once activated, Dendritic Cells (DCs) migrate to the lymphoid organs and exert their role as professional antigen presenting cells. They are able to induce the activation and differentiation of naïve T cells into different types of T helper cells. The T cell response must be suited to the type of inflammation. This is ensured by DCs at two levels. First DCs are functionally plastic. This means that their behavior is subdued to the integrated signals coming from the inflammatory microenvironment. Secondly, the DC population is diverse. Indeed, different DC subsets have different functional specializations. My thesis was focused on the differential response of human DC subsets to Thymic stromal lymphopoietin (TSLP). This cytokine is secreted by inflamed skin and epithelia, and strongly activates myeloid DCs. The TSLP-activated DCs secrete the inflammatory chemokines CCL17 and CCL22, prime an inflammatory Th2 response, and have been involved in the pathogenesis of allergic inflammation. By systematically comparing the response of human blood BDCA-1+ and BDCA-3+ DCs to TSLP stimulation we found that both of these DC subsets get activated by TSLP. However TSLP synergizes with TGF-β to induce the differentiation of blood BDCA-1+ and not BDCA-3+ DCs into Langerhans Cells. Moreover, TSLP induces cell migration and chemokine secretion only on the blood BDCA-1+ subset. Further analysis of the mechanisms implicated in TSLP-induced DC migration revealed that TSLP is required to induce DC migration, but this effect is dependent on the expression of a PTX-sensitive chemokine receptor. Overall our results reveal new precursor capacities of blood DC subsets, different functional properties of blood DC subsets stimulated by TSLP and highlight intricate mechanisms underlying TSLP-induced DC migration.

Este trabalho objetivou avaliar os efeitos imunomoduladores das células-tronco imaturas da polpa dentária (CTIPD) sobre a diferenciação, maturação das células dendríticas derivadas de monócitos (mo-DC), sua capacidade para ativar linfócitos T (Lin T), bem como verificar fatores solúveis liberados nos cocultivos celulares. As analises foram feitas por citometria de fluxo. Foi observado que mo-DC tiveram a diminuição de moléculas relacionadas à diferenciação para mo-DC e o aumento de moléculas relacionados ao estado precursor, o que parece ter refletido na maturação para mDC, verificado pela diminuição das moléculas de maturação. As mo-DC tiverem sua função em induzir a proliferação de Lin T reduzida, além, de favorecer o aumento da proporção de Lin T CD4+FoxP3+IL-10+ e Lin T CD4+FoxP3+IFN-<font face=\"Symbol\">g+. A mensuração dos fatores solúveis dos cocultivos mostrou que houve aumento de fatores anti-inflamatórios e redução de fatores pró-inflamatórios. Futura investigações podem suportar o uso das CTIPD em uma abordagem imunomoduladora utilizando DC em aplicações clínicas. / This study aimed to evaluate the immunomodulatory effects of immature stem cells from dental pulp on differentiation, maturation of monocyte-derived dendritic cells (mo-DC), their ability to activate T cells, as well as identify soluble factors released in cellular cocultures. The analyzes were performed by flow cytometry. It was observed a decrease of molecules related to mo-DC differentiation and an increase of molecules related to precursor state, which seems to have reflected to mDC maturation verified by reduction of maturation molecules. The mo-DC have their role in inducing T cells proliferation reduced, in addition, favored increase CD4+FoxP3+IL-10+ and CD4+FoxP3+IFN-<font face=\"Symbol\">g+ T cells population proportion. The measurement of soluble factors from coculture showed an increase of anti-inflammatory factors and a reduction of pro-inflammatory factors. Future research may support the use immature stem cells from dental pulp in an immunomodulatory approach using DC in clinical applications.

A cromoblastomicose é uma micose subcutânea, com alto índice de morbidade, sendo Fonsecaea pedrosoi (F. pedrosoi) considerado o maior agente etiológico dessa micose, caracterizando uma doença crônica, geralmente confinada na pele e tecidos subcutâneos. Raramente os indivíduos apresentam cura dessa doença, pois as terapias contemporâneas mostram-se deficientes e poucos trabalhos relatam a relação parasito-hospedeiro. As células dendríticas (DCs) são especializadas na apresentação de antígenos para linfócitos T naive induzindo respostas imunes primárias. Diante disso, propomos estudar a capacidade migratória de DCs após infecção com conídios de F. pedrosoi, uma vez que o processo de migração dessas células está intimamente ligado com a sua função sobre as células T, levando ao desenvolvimento de uma resposta imune adaptativa protetora. O fenótipo de DCs foi avaliado através de células obtidas dos linfonodos poplíteos, inguinais e patas de camundongos BALB/c após 12, 24 e 72 horas de infecção com conídios do fungo. Células obtidas foram marcadas com anticorpos específicos e analisadas por citometria de fluxo. Após 24 e 72 horas de infecção verificamos uma diminuição significativa na porcentagem de DCs nas patas, e um aumento significativo dessas células nos linfonodos após 72 horas. A expressão de marcadores de superfície como CCR7 e moléculas co-estimulatórias, mostraram-se diminuídas nas células obtidas das patas. Como no processo de imunofenotipagem podemos analisar DCs vindas de diversos locais, para melhor avaliar a capacidade migratória das DCs, células das patas foram marcadas in vivo injetando-se subcutaneamente o corante CFSE juntamente com conídios do fungo. Verificamos que após 12 e 72 horas, as DCs das patas dos animais infectados, migraram para os linfonodos regionais. Assim constatamos que o fungo F. pedrosoi é capaz de induzir a migração de macrófagos e neutrófilos para o sítio de infecção em poucas horas, leva ao aumento de células B nos linfonodos após 12 e 72 horas de infecção, e também leva ao aumento de linfócitos T CD4+ tanto no local de infecção quanto nos linfonodos. Os resultados também comprovam que o fungo F. pedrosoi foi capaz de ativar as DCs, induzindo sua migração para os linfonodos regionais. / The chromoblastomycosis is a subcutaneous mycosis with a high morbidity rate, Fonsecaea pedrosoi (F. Pedrosoi) being the largest etiologic agent of this mycosis, featuring a chronic disease, usually confined to the skin and subcutaneous tissues. Rarely do people have cure for this disease, because the therapies shown to be deficient contemporary and few studies report the host-parasite relationship. Dendritic cells (DCs) are specialized in presenting antigens to naïve T lymphocytes inducing primary immune responses. Therefore, we propose to study the migratory capacity of DCs after infection with conidia of F. pedrosoi, since the migration of these cells is intimately linked to its function on T cells, leading to development of a protective adaptive immune response. The phenotype of DCs was evaluated using cells obtained from popliteal lymph nodes, sub cutaneous tissue of BALB/c mice after 12, 24 and 72 hours of infection with conidia of the fungus. Cells were labeled with specific antibodies and analyzed by flow cytometry. After 24 and 72 hours of infection we found a significant decrease in the percentage of DCs in the sub cutaneous tissue, and a significant increase of these cells in the lymph nodes after 72 hours. The expression of surface markers such as CCR7 and costimulatory molecules, were reduced in cells obtained from the sub cutaneous tissue. Like the process of immunophenotyping we can analyze DCs coming from various locations, to better assess the migratory capacity of DCs, cells were stained paws in vivo by injecting dye subcutaneously with CFSE conidia of the fungus. We found that after 12 and 72 hours, DCs sub cutaneous tissue of infected animals migrated to regional lymph nodes. We found that the fungus F. pedrosoi is capable of inducing migration of macrophages and neutrophils to the site of infection within a few hours, leads to increased B-cells in lymph nodes after 12 and 72 hours of infection, and also leads to an increase of CD4 + T lymphocytes in both the site of infection and in the lymph nodes. The results also show that the fungus F. pedrosoi was able to activate the DCs, inducing their migration to regional lymph nodes.

Resultados anteriores do nosso grupo mostraram que a melatonina (MLT) age em monócitos aumentando sua sensibilidade à IL-4 e, consequentemente, levando estas células a se diferenciarem em células dendríticas - quando in vitro e estimulados com IL-4 e GM-CSF (mo-DCs) com um perfil fenotípico e funcional mais ativado. Outros estudos do nosso grupo mostraram que mo-DCs de pacientes portadoras de câncer apresentam desvios funcionais capazes de comprometer a ativação de linfócitos por este tipo celular. Um outro apontamento de nosso grupo foi para o fato de que, muito provavelmente relacionado a estes desvios, está um comprometimento da via IL-4/STAT-6/SOCS-5, que se mostrou alterada em pacientes com leucemia linfóide crônica. Baseado nestes resultados, o presente trabalho objetivou investigar os efeitos da MLT na diferenciação in vitro de mo-DCs de doadoras saudáveis e de pacientes portadoras de câncer de mama, sob a hipótese de que este hormônio poderia agir na via IL-4/STAT-6/SOCS-5 de modo a gerar mo-DCs com fenótipo e função relacionados à maior ativação. Para tanto, monócitos provenientes do sangue periférico de indivíduos saudáveis e de pacientes foram tratados com MLT (2,5 nM 2 horas) e induzidos à diferenciação em mo-DCs; no quinto dia as células foram ativadas com LPS (100 ng/ml 24 horas). As análises, por citometria de fluxo, do fenótipo e das citocinas ao longo da diferenciação de mo-DCs não apontou efeitos consistentes acerca do tratamento com MLT, mas indicou maior expressão de CD83+ nos monócitos das pacientes (p=0,014) e maior concentração da citocina IL-12p70 no sobrenadante das culturas de mo-DCs destes indivíduos, ao final da diferenciação (p=0,02). Para a análise da capacidade linfoestimuladora das mo-DCs foi realizada co-cultura destas células com linfócitos T (LT) alogeneicos (1 DC: 30 LT) por 5 dias. Não houve diferença na indução de proliferação de LT estimulados por mo-DCs de saudáveis nem de pacientes. A MLT mostrou efeito apenas nas co-culturas com células saudáveis aumentando as concentrações de TNF, IFN-&#947; e IL-2 nos sobrenadantes. Nas co-culturas com células de pacientes sem tratamento, houve maior nível de IL-2 e IL-10, se comparadas com saudáveis. Os monócitos foram também tratados com MLT (2.5 nM) ou IL-4 (50 ng/mL) por 15 minutos, para avaliação da expressão de STAT-6, pSTAT-6 e SOCS-5. Não foi constatado efeito da MLT nesse caso, mas os monócitos de pacientes apresentaram maior expressão de STAT-6, porém menor de pSTAT-6, comparados com saudáveis. Tomados em conjunto, os resultados globais indicam algumas diferenças fenotípicas e funcionais entre monócitos de pacientes e controles, mas não entre suas mo-DCs. Quanto à MLT, os resultados apontam para o fato de que o hormônio gera alterações apenas em células provenientes de indivíduos saudáveis e somente com relação às citocinas encontradas nos sobrenadantes das co-culturas. / Previous studies at our lab have shown that melatonin (MLT) acts on monocytes increasing their sensitivity to Interleukin-4 (IL-4) and, consequently, generating dendritic cells (DCs) when in vitro and treated with IL-4 and GM-CSF (mo-DCs) phenotically and functionally more activated. Other studies from our group have shown that mo-DCs obtained from cancer patients have functional biases capable of compromising the activation of T lymphocytes. Another result from our group pointed to the fact that part of the mo-DCs\' functional bias in cancer patients could be attributed to the decrease in STAT-6 signaling, a pathway that is activated by IL-4 and down regulated by SOCS-5, whose levels, in turn, were found elevated in cancer patients\' monocytes. Based on these results, the present work aimed to investigate the effects of MLT on the in vitro differentiation of healthy donors and breast cancer patients mo-DCs, under the hypothesis that this hormone could act on the IL-4/STAT-6/SOCS-5 axis generating better activated mo-DCs. Peripheral blood monocytes from healthy donors and breast cancer patients were treated with MLT (2,5 nM 2 hours) and induced to differentiate into mo-DCs; on the fifth day cells were activated with LPS (100 ng/ml 24 hours). Flow cytometry analysis of phenotype and cytokines in supernatants during mo-DCs differentiation didnt show MLT effects, but indicated higher frequency of CD83+ (p=0,014) monocytes among mononuclear cells of the patients, when compared with healthy donors. In addition, higher concentration of IL-12p70 was found in mo-DCs cultures (sixth day - p=0,02). The capacity to stimulate allogeneic T lymphocytes was assessed by co-cultures (1DC : 30LT), maintained for 5 days. Results indicate an effect of MLT only in order to increase the TNF, IFN-&#947; and IL-2 concentrations in supernatants of co-cultures with healthy donors mo-DCs. Patients cells showed differences only when there was no hormone treatment, showing higher levels of IL-10 in the co-culture supernatants, when compared to healthy controls. Monocytes were also treated with MLT (2,5 nM) or IL-4 (50 ng/mL) for 15 minutes to evaluate STAT-6, pSTAT-6 and SOCS-5 expression. Results showed an increase of STAT-6 in patients monocytes and a lower capacity of these cells to phosphorylate this molecule, even when in presence of IL-4. Together, the results point to some phenotypic and functional differences between patients and healthy donors monocytes, but it was not shown in their mo-DCs. Results also point to the fact that MLT generates changes only in healthy donors cells and those effects were only seen with cytokines found in cultures supernatants.

Les cellules dendritiques (DC), principales cellules présentatrices d’antigène, jouent un rôle crucial dans la régulation de la réponse immunitaire. Leur état d’activation et de maturation conditionne notamment la réponse immunitaire anti-tumorale. Ainsi à un stade immature, les DC sont incapables d’activer la réponse immunitaire. Notre équipe a montré qu’en plus d’être inefficaces, les DC intra-tumorales acquièrent des propriétés immunosuppressives et inhibent l’activation lymphocytaire. A l’inverse, activées et matures, les DC peuvent être utilisées en immunothérapie des cancers. Notre équipe a montré que les DC peuvent dans certaines conditions devenir tumoricides et que ces DC tumoricides combinent une activité cytotoxique contre les cellules tumorales et la capacité d’activation des lymphocytes T spécifiques de la tumeur. Avant d’utiliser ces DC tumoricides en phase clinique, nous avons voulu étudier les interactions de ces cellules avec des cellules immunosuppressives générées par les tumeurs, les lymphocytes T régulateurs (Treg). Notre étude montre que les DC cytotoxiques (KDC), générées à partir du sang de patients atteints de tumeurs, sont capables d’inhiber la génération des lymphocytes T régulateurs, impliqués dans l’immunosuppression induite par les tumeurs. Elles sont également à l’origine de la polarisation des lymphocytes T naïfs en des lymphocytes T helper 1, principaux acteurs de la réponse immunitaire anti-tumorale. Sans parvenir à identifier les molécules impliquées, nous avons établi que l’inhibition de la génération des Treg par les KDC n’implique ni l’IL-6, ni le NO, mais est partiellement dépendante d’un contact cellulaire. La rate joue un rôle central dans les mécanismes de tolérance immunitaire, lors des cancers, mais également lors des maladies auto-immunes (MAI). Une meilleure compréhension de la réponse immunitaire au cours de ces 2 types de pathologies au niveau d’un organe lymphoïde majeur tel que la rate est indispensable pour adapter au mieux les thérapeutiques. Cependant, il n’existe que très peu d’études portant sur les DC spléniques humaines. Nos travaux sur les DC au sein de rates provenant de patients atteints de divers cancers et MAI mettent en évidence une répartition des sous populations de DC qui diffère selon les pathologies. Bien que limité par le nombre d’échantillons humains, nous avons également déterminé le profil d’activation de ces sous populations de DC après stimulation par différents TLR. Etant donné le rôle clé des DC dans l’initiation de la réponse immunitaire spécifique, ces nouvelles connaissances pourraient permettre de cibler certains sous-types de DC afin de les activer ou de les inhiber lors de ces différentes pathologies. Ces observations offrent d’importantes perspectives pour la future utilisation des DC dans les stratégies d'immunothérapie. / Known for years as professional antigen presenting cells (APC), dendritic cells (DC) play a crucial role in immune response regulation. Their state of activation and maturation conditions the antitumor immune response. Thus, at an immature stage, the DC are unable to activate the immune response. Our team demonstrated that besides being ineffective, intra-tumoral DC acquire immunosuppressive properties and inhibit T cell activation. Conversely, activated and mature DC can be used in cancer immunotherapy. Previous reports from our research team deciphered that under certain conditions DC can become tumoricidal. These DC combine a cytotoxic activity against tumor cells and the capacity to activate tumor specific T cells. Prior launching the possibility of the application of such cytotoxic DC (referred as KDC for Killer DC) in clinic, we explored the interactions of these cells with regulatory T cells (Treg), -major immunosuppressive cells promoted by tumor microenvironment. Our study revealed that KDC, generated from the blood of cancer patients, can inhibit Treg generation and deviate naive T cell polarization into T helper 1 cells, key players in the anti-tumor immune response. We have further established that the inhibition of Treg generation by the KDC requires cell to cell contact, though implies neither IL-6 nor NO, but the mechanism still to be deciphered. Spleen, a major lymphoid organ, drives foremost mechanisms in immune tolerance during cancer and autoimmune diseases (AID). A better understanding of the immune response specifications during these 2 types of diseases in the spleen is therefore essential goal for therapeutic developments. Nevertheless, there is negligible volume of studies on human splenic immune cells, in particular DC. Our research on splenic DC, derived from patients with various cancers and AID, display distinct DC subset distribution respective to the disease. Although limited by the availability of human splenic samples, we also determined the activation profile of DC subsets after stimulation by different TLR ligands. Given the key role of DC in initiating specific immune response, this novel findings could be implemented to target certain DC subset activation or inhibition in these pathologies. These investigations provide important knowledge for more refine exploitation of DC in immunotherapeutic strategies as treatment tools or as targets.

A asma afeta aproximadamente 300 milhões de pessoas no mundo e é a maior causa de internação hospitalar em crianças nos países desenvolvidos. Essa doença é incurável e por vezes refratária ao tratamento em um número significativo de pacientes. As taxas de prevalência de tabagismo entre pacientes asmáticos são semelhantes aos da população em geral e o impacto da fumaça de cigarro nestes pacientes ainda é clinicamente controverso. O objetivo deste trabalho foi traçar o perfil inflamatório e analisar o papel das células dendríticas sobre a imunomodulação provocada pela exposição à fumaça do cigarro na inflamação alérgica pulmonar induzida previamente por ovalbumina (OVA) em um modelo murino. Primeiramente avaliamos in vivo a ação da fumaça de cigarro na inflamação pulmonar alérgica crônica avaliando a responsividade brônquica, o remodelamento pulmonar, a produção de anticorpos antígeno-específicos, o perfil de células inflamatórias pulmonares e sistêmicas e a produção de citocinas inflamatórias e moduladoras. Em seguida, realizamos estudo in vitro do perfil de maturação, migração e inflamatório de células dendríticas expostas a OVA e/ou a extrato de fumaça de cigarro. Nosso estudo mostrou que a sensibilização e desafios inalatórios com OVA levaram à inflamação pulmonar de característica Th2 com aumento de responsividade brônquica, remodelamento, altos níveis de IgE e de citocinas pró-inflamatórias como IL-4, IL-5 e IL-13. A exposição à fumaça de cigarro, surpreendentemente, levou a uma redução dos níveis de IL-4, IL-5 e IL-13, e simultaneamente reduziu os níveis de citocinas anti-inflamatórias como IL- 10 e TGF-beta em animais sensibilizados e desafiados com antígeno. Foi observada nestes animais, uma redução no número de eosinófilos no lavado broncoalveolar e aumento no número de neutrófilos no pulmão. A combinação da inflamação alérgica com exposição à fumaça de cigarro levou a um aumento do recrutamento e ativação de células dendríticas linfoides nos linfonodos mediastinais, que mostrou relação direta com aumento do influxo de células T CD8+ e ativação das mesmas no pulmão. A inflamação alérgica juntamente com a exposição à fumaça de cigarro, levou a uma redução no recrutamento de células dendríticas plasmocitoides além de reduzir o recrutamento de células T regulatórias. In vitro, mostramos que o extrato de fumaça de cigarro combinado ao antígeno aumenta a capacidade migratória e fagocítica do antígeno pelas BMDCs. No entanto, houve redução da expressão gênica para IL-13 neste mesmo grupo. Concluímos que neste modelo de inflamação pulmonar alérgica crônica combinada com a exposição à fumaça de cigarro leva a uma descaracterização do perfil inflamatório característico da resposta Th2 com a redução do recrutamento de eosinófilos, redução dos níveis de IL-4, IL-5 e IL-13 aliados a um aumento do número de neutrófilos, o que pode estar relacionado ao aumento do recrutamento e ativação de células dendríticas linfoides bem como de células T CD8+ e redução local de células dendríticas plasmocitoides. Mostramos ainda que a fumaça de cigarro juntamente com o antígeno leva as células dendríticas a aumentarem sua capacidade fagocítica porém, reduzir sua capacidade pró-inflamatória pela expressão gênica reduzida de IL-13 / Asthma affects approximately 300 million people worldwide and it is the major cause of hospitalization among children in developed countries. This disease is often refractory to treatment in a high number of patients. The prevalence rates of smoking among asthmatic patients are similar to the general population and the impact of cigarette smoke is also clinically controversial. The main goal of this study is to outline, in a murine model, the inflammatory profile and to analyze the role of dendritic cells on the immunomodulation caused by exposure to cigarette smoke in ovalbumin (OVA)-induced pulmonary allergic inflammation. First, we evaluated in vivo the action of cigarette smoke on chronic allergic pulmonary inflammation, evaluating the bronchial responsiveness, pulmonary remodeling, the production of antigen-specific antibodies, pulmonary and systemic inflammatory cell profile and the production of inflammatory and modulating cytokines. Next, we performed an in vitro study of the maturation, migration and inflammatory profile of dendritic cells exposed to OVA and/or cigarette smoke extract. Our study showed that sensitization and challenge with OVA led to Th2-type lung inflammation with increased bronchial responsiveness, remodeling, high levels of IgE and proinflammatory cytokines such as IL-4, IL-5 and IL-13. Exposure to cigarette smoke has surprisingly led to a reduction in levels of IL-4, IL-5 and IL-13, and simultaneously reduced levels of anti-inflammatory cytokines such as IL-10 and TGF-beta in animals sensitized and challenged with the antigen. We also observed a reduction in the number of eosinophils in bronchoalveolar lavage fluid and an increase in the number of neutrophils in the lung of these animals. The combination of allergic inflammation with exposure to cigarette smoke led to increased recruitment and activation of lymphoid dendritic cells in the mediastinal lymph nodes, which showed to be direct related with increased activation and influx of CD8+ T cells in lung. Allergic inflammation combined with cigarette smoke led to a decrease of plasmacytoid dendritic cells a well as regulatory T cells. In vitro, we showed that cigarette smoke extract combined with antigen increased migratory and phagocytic capacity of BMDCs. However, there was a reduction of IL-13 gene expression in this same group. We conclude that in this model of chronic pulmonary allergic inflammation combined with exposure to cigarette smoke leads to a mischaracterization of the characteristic inflammatory profile of the Th2 response with the reduction of eosinophil recruitment, reduction of levels of IL-4, IL-5 and IL-13 allied to increased number of neutrophils, which is related to increased recruitment and activation of lymphoid dendritic cells as well as CD8+ T cells and local decrement of plasmacytoid dendritic cells. We further show that cigarette smoke combined with antigen increases dendritic cell phagocytic capacity however, reduces its pro-inflammatory capacity by the reduced gene expression of IL-13

INTRODUÇÃO: Asma é uma doença inflamatória crônica das vias aéreas que envolve diversos tipos de células, especialmente eosinófilos, células T, macrófagos, células epiteliais e células dendríticas. Durante a exposição alérgica, células dendríticas migram para os linfonodos broncopulmonares e iniciam a resposta imune na asma. Em asma humana, poucas informações sobre células dendríticas, células B, células T, eosinófilos, VCAM em linfonodos broncopulmonares são conhecidas. Poucos estudos também descrevem a interação celular entre linfonodos e vias aéreas na asma durante exacerbações. MÉTODOS: Foram analisados por método histoquímico, imuno-histoquímico e análise de imagens as expressões de Vermelho Congo, FatorXIIIa+, CD83+, CD207+, CD1a+, CD23+, CD20+, CD4+, CD8+, VCAM, em vias aéreas grandes e linfonodos broncopulmonares de 11 indivíduosnão asmáticos falecidos por asma e 8 controles não asmáticos. A análise dos marcadores foi realizada na região cortical dos linfonodos e em três regiões das vias aéreas: camadas interna, muscular e externa. RESULTADOS: Os indivíduos asmáticos apresentaram maior expressão de eosinófilos nos lifonodos broncopulmonares e nas três camadas das vias aéreas. Os marcadores FatorXIIIa+, CD23+, CD20+, CD4+ e CD8+ apresentaram aumento na camada externa das vias aéreas dos indivíduos asmáticos. CONCLUSÕES: Os eosinófilos estão aumentados nos linfonodos broncopulmonares e nas vias aéreas dos asmáticos. Alguns marcadores como o FatorXIIIa+, CD20+, CD4+, CD8+ e CD23+ estão aumentados apenas na camada externa das vias aéreas dos asmáticos. VCAM, CD83+, CD207+, CD1a+ não apresentaram aumento nos asmáticos. Este resultado sugere que esses marcadores não estão relacionados ao evento da asma fatal nos indivíduos estudados. As correlações encontradas entre vias aéreas e linfonodos nos asmáticos sugerem que na asma fatal ocorra um fluxo celular direcionado. Nossos resultados fornecem novas evidências para a participação do linfonodo broncopulmonar na exacerbação da asma / INTRODUCTION: Asthma is a chronic inflammatory disease of the airways that involves many different cells, specially mast cells, eosinophils, T cells, macrophages, epithelial cells and dendritic cells (DCs). During allergen exposure, pulmonary DCs migrate to bronchopulmonary lymph nodes (LNs) and prime the immune cells that will characterize the immune response in asthma. In human asthma, there is no information about the composition of DCs, B cells, T cells, and vessels in the regional LNs involved in the immune responses to inhaled antigens. Also, there is little information about the lung - LN cells trafficking occurring in asthma during exacerbations. METHODS: Using histochemistry, immunohistochemistry and image analysis, we investigated the expression of Congo Red+ (eosinophil), factor XIIIa+, CD23+, CD4+, CD8+, CD20+, CD207+, CD83+, CD1a+ cells and VCAM-1+ in the large airways and bronchopulmonary lymph nodes of 11 non-smoker patients that died due to an asthma exacerbation and compared with 8 deceased non-asthmatic controls. The analysis of the markers was carried out in the cortical área of the lymph nodes and three layers of the airways: internal, airway smooth muscle and outer layer. RESULTS: The LNs of asthmatics had increased expression of eosinophils when compared to controls. The large airways of asthmatics had increased expression of eosinophils in all the layers and factor XIIIa+, CD4+, CD8+, CD20+ and CD23+ had increased in the outer layer. CONCLUSIONS: A fatal asthma episode is associated with an altered expression of eosinophils in LNs and large airways.Factor XIIIa+ monocyte dendritic cel, CD4+, CD8+, CD20+, CD23+ cells had increased in the large airways without a concomitant increase in the expression of these cells in bronchopulmonary LNs.However, some DC cell trafficking between the airway mucosa and LNs seems occurs in this severe fatal asthma exacerbation