Orientador: Edson Arantes / Tese (doutorado) - Universidade Estadual de Campinas, Faculdade de Ciencias Medicas / Made available in DSpace on 2018-08-11T19:45:11Z (GMT). No. of bitstreams: 1 Lintomen_Leticia_D.pdf: 1121941 bytes, checksum: 2c92a8ddfce83c01ed6c71e524096e0b (MD5) Previous issue date: 2008 / Resumo: Os eosinófilos participam da patogênese de várias doenças inflamatórias, incluindo infecções parasíticas e doenças alérgicas. Dentre as doenças alérgicas, a eosinofilia tem presença marcante na asma. Atualmente, a asma afeta 300 milhões de indivíduos no mundo, sendo considerada um problema de saúde pública mundial. A asma é uma doença inflamatória crônica que envolve interações entre fatores externos e genéticos, e tem como características principais a inflamação pulmonar e a hiperresponsividade brônquica. O recrutamento de eosinófilos para as vias aeríferas contribui para o caráter crônico da asma. A migração de eosinófilos para o tecido inflamado é um processo complexo, que é regulado por numerosos fatores, incluindo citocinas, quimiocinas, óxido nítrico (NO) e interações de moléculas de adesão. Estudos prévios investigaram as interações funcionais entre NO e CC-quimiocinas. Young e colaboradores (1999) mostraram que o número de eosinófilos no lavado broncoalveolar (LBA) de macacos desafiados está marcadamente aumentado 24 horas após o desafio, e que este aumento é acompanhado de altos níveis de NO no ar exalado e de eotaxina no LBA. Em pacientes com rinite, a eotaxina aumentou o número de eosinófilos no fluido do lavado nasal e também os níveis de NO nasal (Hanazawa et al., 1999). Em contraste, em modelos murinos de asma a inibição seletiva da NOS induzível resultou na redução da migração eosinofílica para os pulmões (Feder et al., 1997; Iijima et al., 2001), que estava associada com o aumento da expressão da CC-quimiocina proteína quimiotática para monócito-1 (MCP-1) no tecido pulmonar (Trifilieff et al., 2000). Além disso, NO (ou doadores de NO) também são capazes de inibir a produção de RANTES (Frank et al., 2000). O NO via formação de peroxinitrito (ONOO-) também pode reduzir a migração de eosinófilos induzida por eotaxina (Sato et al., 2000). Entretanto, o papel modulatório do NO nas funções do eosinófilo mediadas por CC-quimiocinas ainda permanece contraditório. Portanto, o presente trabalho investigou o efeito modulatório do NO na adesão aumentada, quimiotaxia e desgranulação do eosinófilo induzidas pelas Ccquimiocinas eotaxina e RANTES in vitro, e a expressão de VLA-4 e Mac-1 na superfície do eosinófilo. Nós realizamos ensaios funcionais (adesão e desgranulação), análise da expressão de moléculas de adesão por citometria de fluxo (VLA-4 e Mac-1) e a investigação de resíduos de tirosina nitrada para avaliar interações do NO com CC-quimiocinas em eosinófilos humanos. Os ensaios de MTT mostraram que as incubações de eosinófilos por 2, 3, ou 4 horas com eotaxina (10, 100 e 1000 ng/ml) ou RANTES (10, 100 e 1000 ng/ml) não afetam a viabilidade celular e, em determinadas condições, até promovem a ativação das células. Os resultados de adesão à fibronectina mostraram que a eotaxina (10, 100 e 1000 ng/ml) ou RANTES (10, 100 e 1000 ng/ml) não aumentam a adesão de eosinófilos em períodos de incubação de 2 e 3 horas. Entretanto, a incubação de eosinófilos por 4 horas com eotaxina ou RANTES aumentou significativamente a adesão à fibronectina. O L-NAME (0.1 mM), individualmente, aumentou significativamente a adesão de eosinófilos à fibronectina; porém a co-incubação de L-NAME com eotaxina (ou RANTES) não afetou a adesão observada com cada agente isoladamente. Além disso, a expressão de VLA-4 e de Mac-1 não foi modificada em nenhuma das condições experimentais testadas. Eotaxina e RANTES também não foram capazes de aumentar os níveis de GMPc nos eosinófilos, em condições onde o SNP (0.1 mM), usado como controle positivo, aumentou significativamente os níveis desse segundo mensageiro. Além disso, eosinófilos tratados com L-NAME, eotaxina e RANTES, individualmente, foram capazes de desgranular estas células, mas a co-incubação de LNAME com eotaxina (ou RANTES), não alterou esta resposta. Os resultados de quimiotaxia mostraram migração significativa de eosinófilos (tratados ou não com LNAME) em resposta à eotaxina ou RANTES. Os resultados obtidos de Western blotting para 3-nitrotirosina mostraram ausência de proteínas nitradas nos eosinófilos de indivíduos sadios ou asmáticos. No conjunto, nossos resultados mostram que, nas condições experimentais estabelecidas, o NO não modula adesão, migração e desgranulação em eosinófilos estimulados com eotaxina ou RANTES / Abstract: Eosinophils participate in the pathogenesis of many inflammatory diseases, including parasitic infections and allergic diseases. Of the allergic diseases, asthma is characterized by eosinophilia. Currently, asthma affects 300 million people in world, and is an important public health problem. Asthma is an inflammatory chronic disease that involves interactions between external and genetic factors, and has lung inflammation and bronchial hyperresponsiveness as major features. The recruitment of eosinophils into airways contributes to the asthma chronic character. The eosinophil migration to inflamed tissue is a complex process regulated by several factors, including cytokines, chemokines, nitric oxide (NO) and adhesion molecule interactions. Previous studies have investigated the functional interactions between NO and CC-chemokines. Young et al. (1999) showed that the number of eosinophils in the bronchoalveolar lavage (BAL) fluid of challenged monkeys is markedly increased at 24 h post-challenge, accompanied by higher levels of both exhaled NO and eotaxin in BAL fluid. In rhinitis patients, eotaxin increased the number of eosinophils in the nasal lavage fluid, and that was also accompanied by elevated nasal NO levels (Hanazawa et al., 1999). In contrast, in murine models of asthma, selective inhibition of inducible NOS resulted in a reduction in pulmonary eosinophil migration (Feder et al., 1997; Iijima et al., 2001), with an increased expression of the CC-chemokine monocyte chemoattractant protein-1 (MCP-1) in the lung tissue (Trifilieff et al., 2000). Moreover, NO (or NO donors) have also been shown to inhibit the production of RANTES (Frank et al., 2000). Nitric oxide via peroxynitrite (ONOO-) formation is reported to reduce eotaxin-induced eosinophil migration (Sato et al., 2000). However, the modulatory role of NO in the CC-chemokines-mediated eosinophil functions is still not well understood. Therefore, the present study was designed to investigate the modulatory effect of NO in the enhanced eosinophil adhesion, chemotaxis and degranulation induced by the Ccchemokines eotaxin and RANTES in vitro, and the expression of VLA-4 and Mac-1 on the eosinophil surface. We therefore carried out functional assays (adhesion and degranulation), flow cytometry analysis of adhesion molecules (VLA-4 and Mac-1 expression) and investigation of tyrosine nitration to evaluate the interactions between NO and CC-chemokines in human eosinophils. MTT assays showed that incubation of eosinophils for 2, 3 or 4 hours with eotaxin (10, 100 and 1000 ng/ml) or RANTES (10, 100 and 1000 ng/ml) did not affect cellular viability and, in some conditions, even caused cellular activation. The results of adhesion to fibronectin showed that eotaxin (10, 100 and 1000 ng/ml) or RANTES (10, 100 and 1000 ng/ml) did not increase eosinophil adhesion at 2 or 3 hours of incubation. Nevertheless, the incubation of eosinophils for 4 hours with eotaxin or RANTES significantly increased adhesion to fibronectin. L-NAME (0.1 mM), alone, significantly increased eosinophil adhesion to fibronectin; however the co-incubation of L-NAME with eotaxin (or RANTES) did not affect the adhesion of each agent. Moreover, expression of VLA-4 and Mac-1 were not modified by any of the experimental conditions. Eotaxin and RANTES did not increase eosinophil cGMP levels under the same conditions in which SNP (0.1mM), used as a positive control, significantly increased the levels of this second messenger. Furthermore, eosinophils treated with L-NAME, eotaxin and RANTES, alone, demonstrated degranulation, however the co-incubation of eosinophils with L-NAME and eotaxin (or RANTES) did not alter this response. Chemotaxis assays showed a significant eosinophil (treated or not with L-NAME) migration in response to eotaxin or RANTES. Western blotting for 3-nitrotyrosine-3 showed a lack of nitrated proteins in eosinophils from healthy or asthmatic donors. Taken together, results show that, under the experimental conditions established, NO does not modulate adhesion, migration and degranulation in eotaxin or RANTES-stimulated eosinophils / Doutorado / Doutor em Farmacologia

No description available.

The chemokine receptor CCR5 has been implicated in the pathogenesis of prostate cancer (PCa). A novel series of piperazine derivatives have been designed and synthesized as CCR5 antagonists and their activity as inhibitors of PCa cell lines proliferation was explored. A lead compound has been identified which induced 100% inhibition of PCa cell proliferation at micromolar concentrations. Anibamine, the only natural product CCR5 antagonist, was also examined for its anti-proliferative activity and was found to inhibit proliferation of PCa cells at micromolar concentrations as well. The expression of RANTES mRNA was observed in DU-145, M12 and P69 cells via RT-PCR, while the expression of CCR5 mRNA was observed only in M12 cells. A CHO-CCR5 stable cell line was prepared for the CCR5 ligand competition binding assays. Both anibamine and the newly identified lead compound will serve as leads in the development of novel CCR5 antagonists as anti-prostate cancer agents.

The focus of this thesis is the role of dengue virus in the induction of chemokines. Dengue virus (DENV) occurs as four distinct serotypes, called DENV 1,2,3,and 4. Symptomatic DENV infection ranges from a self limited febrile illness, dengue fever (DF), to a more severe disease, dengue hemorrhagic fever/dengue shock syndrome (DHF/DSS). DHF is characterized by increased capillary permeability resulting in decreased plasma volume, which may be accompanied by hemorrhagic manifestations. Many factors including T cell cross reactivity, viral burden, antibody dependent enhancement and induction of chemokines and cytokines have been reported in DHF and may play a role in the pathogenesis of DENV infection. Cytokines have been shown to modulate endothelial cell permeability [1-3]. Recent studies have shown that DENV-infected endothelial cells secrete the chemokine, interleukin (IL)-8 in vitro [4]. In addition, the permeability of an endothelial cell monolayer was found to be increased by interleukin-8 (IL-8) in vitro[5]. This thesis examines the effects of DEN2V infection on the induction of chemokines, and specifically, which DEN2V viral protein(s) are involved in the induction of IL-8. The chemokine induction profile following DEN2V infection was initially assessed in various cell lines that may represent potential targets in vivo, including monocytes, liver cells and endothelial cells. We hypothesized that distinct profiles of chemokine secretion can be induced by DEN2V infection of various cell types in vitro. We found RANTES (Regulated upon Activation, Normal T cell Expressed and Secreted) and IL-8 were induced in two of the five cell lines. DEN2V infection of primary monocyte-derived dendritic cells induced RANTES and IL-8 along with macrophage inflammatory protein-1α (MIP-1α), MIP-1β and monocyte chemoattractant protein-1 (MCP-1) but at an earlier time post infection than in the cell lines. These results showed that DEN2V infection induces distinct chemokine profiles in many cell types. In addition, monocytic-derived DCs can secrete chemokines upon infection with DEN2V. Characterization of the signaling pathways induced by DEN2V revealed that DEN2V induction of chemokines in human embryonic kidney (HEK293A) cells is mainly through the nuclear factor kappaB (NFκB) pathway, as previously reported for endothelial cells and 293T cells [4,6]. Alternatively, the liver cell line (HepG2) activated mainly activator protein (AP)-l. In addition, DENV infection can induce the activation of the interferon-stimulated response element (ISRE) driven promoter. IL-8 has been shown to have multiple effects on the immune system ranging from recruiting cells to the site of infection to countering the antiviral effects of type I interferon (IFN) [7,8]. Previous reports have shown that viral proteins can induce chemokines such as seen with IL-8 induction with the nonstructural protein 5A (NS5A) and core proteins from hepatitis C virus [9,10]. We hypothesized that protein(s) from DENV could induce chemokine production. The expression of DENV proteins was analyzed for effects on IL-8 and RANTES production in HEK293A cells. The effects of viral replication on IL-8 and RANTES induction were also analyzed using a DENV replicon that contains genes for the capsid protein and the nonstructural proteins. Transfection of plasmids expressing NS5 or the DEN2V replicon induced the expression and secretion of IL-8 but not RANTES. We attributed the lack of RANTES induction to the inability of NS5 or the DEN2V replicon to induce transcription from the ISRE driven promoter. We also found that NS5 and the DEN2V replicon induced IL-8 mainly through the CCAAT/enhancer binding protein (c/EBP) and AP-1 pathways. The profile of transcription factor activation is different from what was seen with DENV infection of HEK293A cells and suggests that the transient expression of the NS5 protein and the replication and/or translation of the DEN2V genome use different pathways than viral infection to induce IL-8. In addition, we found that the expression of prM-E, known to produce virus-like particles, could induce IL-8 secretion and activate transcription from the IL-8 promoter. As with the expression of NS5, RANTES was not induced. Analysis of the transcription factors involved in IL-8 induction using luciferase reporter constructs indicated that expression of prM-E induced transcription of IL-8 through NFκB, AP-1 and c/EBP, similar to what was seen with DEN2V infection of HEK293A cells. These results suggest that production of virions or virus-like particles induce IL-8 but that another mechanism in the viral life cycle is responsible for the induction of RANTES expression by DEN2V infection. We were also interested in the effects of drugs that have been used previously to inhibit cytokine or chemokine production on chemokine induction during DEN2V infection. We hypothesized that pharmacological inhibitors of cytokines will inhibit secretion of chemokines in DEN2V infected cells. We found that the pharmacological inhibitors SB203580 and rolipram enhanced chemokine production in a DEN2V infected liver cell line (HepG2), whereas dexamethasone had the same effect in a kidney epithelial cell line (HEK293A). We conclude that drugs that inhibit signaling pathways involved in cytokine production in other experimental systems can have variable effects on chemokine induction in different cell types during DEN2V infection. The data generated in this thesis extend our understanding of how DEN2V manipulates the host cell during viral infection to produce chemokines and perhaps enhance viral propagation and dissemination through the induction of IL-8. In addition, this study provides insight into the variable effects pharmacological drug treatment may have on disease progression during DENV infection. These results increase our understanding of DENV pathogenesis and may be helpful in finding better strategies for treatment and prevention.

Allergic asthma is an immunological disease, mediated by CD4+ Th2 cells, and its prevalence has increased over recent decades. Features of allergic asthma include airway hyperresponsiveness (AHR), airway eosinophilia, excessive airway mucus production, and increased IgE and Th2 cytokine levels. Airway remodeling with pulmonary fibrosis is noted in the progress of asthma. In this thesis, a murine model of allergic asthma was used to investigate the effect of cyclophosphamide (CY) on asthma and the involvement of regulatory T cells (Treg), and the role of Granulocyte-macrophage colony stimulating-factor (GM-CSF) in allergic asthma by using GM-CSF knockout mice. CY is a cytotoxic agent, which paradoxically augments several immune responses. The first part of this thesis was aimed to study the effects of CY in a murine model of allergic airway inflammation. BALB/c mice were immunized with ovalbumin (OVA) on days 0 and 14, and challenged with aerosolized OVA from days 21 to 27. Some mice additionally received CY on days -2 and 12. In the CY-treated animals, pronounced worsening of inflammatory features was noted, including increases in eosinophil infiltration, epithelial thickness, mucus occlusion and eosinophil numbers in bronchoalveolar lavage fluid (BALF). Increased total and OVA-specific serum IgE were also noted in the CY-treated animals. In cell cultures from peritracheal lymph nodes, the Th2 cytokines IL-4 and IL-5 were elevated in animals treated with CY. It was hypothesized that the effects of CY could be caused by reduced immunosuppression mediated by Treg. mRNA expression of the immunosuppressive cytokines IL-10 and TGF-beta was reduced in the lungs of CY-treated mice. The expression of FoxP3, a marker of naturally occurring Treg, was significantly reduced in spleens, thymuses and peritracheal lymph nodes after the second injection of CY, and in the lung tissue after allergen challenge in CY-treated mice. Furthermore, lung IL-10-producing CD4+ T cells and CTLA-4+-bearing CD4+ T cells were reduced after allergen aerosol challenge in CY-treated mice. Thus CY worsened the features of allergic pulmonary inflammation in this model, in association with increased production of IgE and Th2 cytokines. The reduction in expression of FoxP3 and immunosuppressive cytokines by CY suggests that toxicity to Treg may contribute to the increased inflammation. GM-CSF plays a role in the growth, development, and maturation of bone marrow hemopoietic cells into mature blood cells, and has been proposed to be involved in potentiating the function of inflammatory cells in allergic inflammation. In the second part of this thesis, GM-CSF knockout (KO) mice were used to investigate the role of GM-CSF. In allergic KO mice, airway eosinophils were only shown in the perivascular, but not peribronchial areas in the lung, compared to the allergic wild-type (WT) mice in which eosinophil infiltration appeared in both areas. Eosinophil numbers were drastically reduced in the bronchoalveolar lavage fluid (BALF) of KO mice. IL-5 production in the lung tissue and BALF in allergic KO mice was reduced; similar results were also found in peritracheal draining lymph nodes after in vitro stimulation assays. However, IL-4 and IL-13 production, airway hyperresponsiveness (AHR), and serum IgE production were not affected in allergic KO mice. Surprisingly, lung IFN-gamma mRNA and BALF levels were increased in allergic KO mice. Lung mRNA levels of CCR3, a key chemokine receptor on eosinophils, were significantly reduced in allergic KO mice, whereas expression of the chemokines eotaxin and RANTES were at similar levels in allergic KO and WT mice. Lung mRNA levels of the IFN-gamma-inducible chemokines Mig (CXCL9) and IP-10 (CXCL10), which are antagonists of CCR3, and their receptor CXCR3 were increased in allergic KO mice, compared with allergic WT mice. Data obtained from flow cytometry showed more eosinophils survived in the lung of WT mice than KO mice. Another allergy model, a peritoneal allergy model was performed to investigate inflammation in a different model. Leukocyte subpopulations such as neutrophils, eosinophils, macrophages, and lymphocytes were reduced in the peritoneal lavage fluid of allergic KO mice. The findings revealed that GM-CSF is essential for IL-5 production, pulmonary airway eosinophilia and eosinophil survival. In the absence of GM-CSF, over-production of IFN-???? may induce chemokines, including Mig and IP-10, which are antagonists for CCR3 and may reduce airway eosinophil infiltration. In this thesis, a murine model of allergic asthma has been used to obtain novel findings on the regulation of allergic inflammation. The results with CY are relevant to the treatment of asthma patients with CY and other cytotoxic agents. The findings in the GM-CSF KO mice suggest that GM-CSF is a potential therapeutic target in asthma, and that in assessment of new therapeutic agents for asthma, effects on GM-CSF should be considered.

Allergic asthma is an immunological disease, mediated by CD4+ Th2 cells, and its prevalence has increased over recent decades. Features of allergic asthma include airway hyperresponsiveness (AHR), airway eosinophilia, excessive airway mucus production, and increased IgE and Th2 cytokine levels. Airway remodeling with pulmonary fibrosis is noted in the progress of asthma. In this thesis, a murine model of allergic asthma was used to investigate the effect of cyclophosphamide (CY) on asthma and the involvement of regulatory T cells (Treg), and the role of Granulocyte-macrophage colony stimulating-factor (GM-CSF) in allergic asthma by using GM-CSF knockout mice. CY is a cytotoxic agent, which paradoxically augments several immune responses. The first part of this thesis was aimed to study the effects of CY in a murine model of allergic airway inflammation. BALB/c mice were immunized with ovalbumin (OVA) on days 0 and 14, and challenged with aerosolized OVA from days 21 to 27. Some mice additionally received CY on days -2 and 12. In the CY-treated animals, pronounced worsening of inflammatory features was noted, including increases in eosinophil infiltration, epithelial thickness, mucus occlusion and eosinophil numbers in bronchoalveolar lavage fluid (BALF). Increased total and OVA-specific serum IgE were also noted in the CY-treated animals. In cell cultures from peritracheal lymph nodes, the Th2 cytokines IL-4 and IL-5 were elevated in animals treated with CY. It was hypothesized that the effects of CY could be caused by reduced immunosuppression mediated by Treg. mRNA expression of the immunosuppressive cytokines IL-10 and TGF-beta was reduced in the lungs of CY-treated mice. The expression of FoxP3, a marker of naturally occurring Treg, was significantly reduced in spleens, thymuses and peritracheal lymph nodes after the second injection of CY, and in the lung tissue after allergen challenge in CY-treated mice. Furthermore, lung IL-10-producing CD4+ T cells and CTLA-4+-bearing CD4+ T cells were reduced after allergen aerosol challenge in CY-treated mice. Thus CY worsened the features of allergic pulmonary inflammation in this model, in association with increased production of IgE and Th2 cytokines. The reduction in expression of FoxP3 and immunosuppressive cytokines by CY suggests that toxicity to Treg may contribute to the increased inflammation. GM-CSF plays a role in the growth, development, and maturation of bone marrow hemopoietic cells into mature blood cells, and has been proposed to be involved in potentiating the function of inflammatory cells in allergic inflammation. In the second part of this thesis, GM-CSF knockout (KO) mice were used to investigate the role of GM-CSF. In allergic KO mice, airway eosinophils were only shown in the perivascular, but not peribronchial areas in the lung, compared to the allergic wild-type (WT) mice in which eosinophil infiltration appeared in both areas. Eosinophil numbers were drastically reduced in the bronchoalveolar lavage fluid (BALF) of KO mice. IL-5 production in the lung tissue and BALF in allergic KO mice was reduced; similar results were also found in peritracheal draining lymph nodes after in vitro stimulation assays. However, IL-4 and IL-13 production, airway hyperresponsiveness (AHR), and serum IgE production were not affected in allergic KO mice. Surprisingly, lung IFN-gamma mRNA and BALF levels were increased in allergic KO mice. Lung mRNA levels of CCR3, a key chemokine receptor on eosinophils, were significantly reduced in allergic KO mice, whereas expression of the chemokines eotaxin and RANTES were at similar levels in allergic KO and WT mice. Lung mRNA levels of the IFN-gamma-inducible chemokines Mig (CXCL9) and IP-10 (CXCL10), which are antagonists of CCR3, and their receptor CXCR3 were increased in allergic KO mice, compared with allergic WT mice. Data obtained from flow cytometry showed more eosinophils survived in the lung of WT mice than KO mice. Another allergy model, a peritoneal allergy model was performed to investigate inflammation in a different model. Leukocyte subpopulations such as neutrophils, eosinophils, macrophages, and lymphocytes were reduced in the peritoneal lavage fluid of allergic KO mice. The findings revealed that GM-CSF is essential for IL-5 production, pulmonary airway eosinophilia and eosinophil survival. In the absence of GM-CSF, over-production of IFN-???? may induce chemokines, including Mig and IP-10, which are antagonists for CCR3 and may reduce airway eosinophil infiltration. In this thesis, a murine model of allergic asthma has been used to obtain novel findings on the regulation of allergic inflammation. The results with CY are relevant to the treatment of asthma patients with CY and other cytotoxic agents. The findings in the GM-CSF KO mice suggest that GM-CSF is a potential therapeutic target in asthma, and that in assessment of new therapeutic agents for asthma, effects on GM-CSF should be considered.

No description available.

Les épithéliums muqueux se présentent comme porte d’entrée majeure du virus de l’immunodéficience humaine (VIH) et jouent un rôle critique dans la transmission sexuelle du virus. Les cellules épithéliales et les cellules dendritiques dans les muqueuses pluristratifiés, sont des cibles initiales pour la transmission virale. Il a été déjà montré, sur les muqueuses génitales chez l’homme, que la circoncision réduit plus de 60% acquisition du virus chez l’homme. Les mécanismes d’entrée du VIH au niveau du tractus génital chez l’homme sont tres mal connus et pratiquement pas étudiés. Nous avons émis l’hypothèse selon laquelle le prépuce, qui est enlevé lors de la circoncision, pourrait être une porte d’entrée majeure du virus. Nous avons d’abord montré que le VIH peut pénétrer effectivement au niveau du prépuce ex vivo, la partie interne du prépuce présente plus permissive que la partie externe. De plus, le virus pénètre uniquement dans le prépuce après formation d’une synapse virologique entre la cellule infectée, présente dans les secrétions génitales et la surface muqueuse du prépuce. La formation de la synapse virologique induit le bourgeonnement massif et polarisé de virions dans la fente synaptique; ceux ci pénètrent dans l’épiderme du prépuce. À l’inverse, l’entrée du VIH sous forme de virus libre dans le prépuce est inefficace. Nous avons ensuite caractérisé la séquence des événements initiaux mis en place lors de l’entrée du VIH dans le prépuce interne. La première cellule cible immune est la cellule de Langerhans (LCs), dont le rôle physiologique est de protéger la muqueuse contre les pathogènes qui l’envahissent. Après la capture du virus les LCs migrent vers le derme pour former des conjugués cellulaires avec les cellules T CD4+ du derme. La dynamique de migration de LCs est régulée par de sécrétion de cytokines et chimiokines (RANTEs et MIP- 3alpha) induites par la pénétration du virus dans le tissu. Nous avons ensuite caractérisé les mécanismes de l’immunité mis en jeu au niveau des kératinocytes de l’épiderme lors de la formation de la synapse virologique et évalué comment ces signaux contribuent à l’entrée efficace du virus dans la muqueuse du prépuce interne chez l’homme, ex vivo. À l’aide d’un modèle cellulaire de muqueuse simplifiée basé sur des kératinocytes primaires humains ex vivo, nous avons montré que les protéines d’enveloppe du VIH , gp120 mais pas gp41, et les molécules d’adhésion (intégrines LFA-1, leurs ligands ICAM-1 and -3) contrôlent la formation de la synapse virologique. La synapse virologique induit au niveau des kératinocytes l’activation de la voie NF- B indépendentmmment de MyD88 après activation du Toll-like-receptor 4 (TLR4) exprimé par les kératinocytes. En recherchant quelle cytokine pouvait être induite par cette signalisation, nous avons montré que la synapse virologique induit la sécrétion par les kératinocytes d’une cytokine produite par les cellules non-hématopoïétiques, la thymic stromal lymphopoietin (TSLP). La TSLP est un chemoattracteur des cellules myéloïdes comme les cellules LCs et peut induire leur maturation. Nous avons ensuite montré que, sur des explants de prépuce humain ex vivo, la synapse virologique induit bien la sécrétion de TSLP, laquelle en premier lieu attire les LCs qui expriment le TSLP-récepteur vers la surface du tissus induisant leur maturation. Les LCs peuvent ensuite migrer dans l’autre sens vers le derme. La sécrétion des cytokines comme RANTEs, MIP-3alpha, qui sont impliquées lors de la pénétration effective du virus dans les tissus n’a pas de rôle dans la signalisation induite par la formation de la synapse virologique proprement dite. Les kératinocytes de l’épiderme du prépuce interne, grâce à leur réponse innée TSLP induite par la synapse virologique ont un rôle déterminant dans l’entrée du VIH dans le prépuce. Ainsi, en réponse de la synapse virologique, les kératinocytes secrètent du TSLP après activation de la voie de NF- B dépendante de MyD88. (...) / The mucosal epitheliums are presented as a major portal of HIV-1, and play a critcal role for the sexual transmission of HIV-1. Epithelial cells, and dendritic cells in the pluristratified mucosa, are the initial targets of viral transmission. It has already been shown, in the genital mucosa in men, circumcision reduces by more than 60 % of virus acqusition by men. The entry mechanisms of HIV-1 in the male urogenital tracts are poorly understood and not pratically studied. We suggested that foreskin, removal by circumcision surgery, could be a major portal of HIV-1 entry. We first showed that HIV-1 could enter efficiently ex vivo inner foreskin mucosa, the inner part of foreskin which is more permissive than the outer part. More over, virus penetrates only after the viral synapse (VS) formation between HIV-1-infected cells and foreskin epidermis, in the presence of genital secretion on the surface of foreskin mucosa. The formation of VS induces massive and polarized budding of HIV-1 particles within the synaptic cleft. The virions therefore penetrate into the foreskin epidermis. Inversely, cell-free virus entry is inefficient in the foreskin. Next, we characterized the initial events of HIV-1 VS formation in the inner foreskin. The first cell type that HIV-1 encounters is Langerhans cells (LCs), whose physiological role is to protect the mucosa against invasive pathogens. After capture of virus, LC migrates towards the dermis to form intercellular conjugates with dermal CD4+ T cells. The dynamic of LC migraiton is regulated by the secretion of cytokines in the mucosa, induced by HIV-1 entry, including RANTEs and MIP-3alpha. We then characterized the mechanisms of foreskin epidermal keratinocyte (KC), activated innate immunity during the VS formation and the signaling pathway contributed to the efficient entry of HIV-1 via inner foreskin mucosa. Using a simplified mucosal model based on human primary foreskin keratinocytes, we demonstrated that HIV-1 envelope protein, gp120, but not gp41 and the adhesion molecules (integrins LFA-1, ICAM-1/-3), contribute to the VS formation. VS induces at the KC level the activation of NF- B pathway by I B and p65 molecules, after activation of TLR4 expressed on KCs. By searching for which cytokine could be induced by this signalisation, we then showed that VS induced the secretion by KC of one cytokine, which is produced by the non-hematopoetic cells, thymic stromal lymphopoietin (TSLP). TSLP is an chemoattractant for myeloid cells like LCs, and could induce LC maturation. We then showed, using the foreskin explants that, VS induces the secretion of TSLP, which first attracts LC expressing constitutively TSLP receptors to the apical surface of foreskin, inducing its maturation. These matured LCs then migrate to another direction towards the dermis. The secretion of cytokines such as RANTEs and MIP-3alpha, involved in the HIV-1 efficient entry has no roles in the signaling induced by VS formation as mentioned above. The Inner foreskin keratinocytes, due to the innate response of TSLP induced by VS, have a determinant role in the HIV-1 entry into the foreskin. Likewise, in response to VS, KCs secrete TSLP after NF- B activation dependent on regulator MyD88. The secreted TSLP, in addition to four different proinflammatory cytokines and chemokines (IL-6, IL-8, MIG, and MMP-9) allows virus to attract LCs to the foreskin surface, to capture the virus present in the synaptic cleft in order to facilite efficient transmission at the level of foreskin mucosa. Likely to the conventional immune cells, KCs, in one hand protects the foreskin mucosa and in another, is hijacked to facilitate HIV-1 transmission.