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Les interférons de type I (IFN-I) peuvent être produits par toutes les cellules mais les rares cellules dendritiques plasmacytoïdes en sont les principales cellules productrices. L’IFNa orchestre de nombreux mécanismes pathogéniques dans l’infection par le virus de l’immunodéficience humaine de type 1 (VIH-1). L’étude de modèles physiologiques et pathophysiologiques peut fournir des informations cruciales sur les moyens d’exploiter la signalisation de l’IFNa dans un but thérapeutique. Les pDCs de femmes produisent plus d’IFNa en réponse à la stimulation du récepteur Toll-like 7 (TLR7) que les pDCs d’hommes. Les mécanismes impliqués dans cette différence n’ont été que partiellement identifiés. Nous démontrons ici un mécanisme par lequel la plus forte expression d’IRF5 dans les pDCs chez les sujets sains féminins, sous le contrôle d’ERa, participe à leur production plus élevée d’IFNa en réponse à TLR7. La co-infection par le virus de l’hépatite C (VHC) est aujourd’hui l’une des principales causes de mortalité parmi les individus infectés par le VIH-1. Nous avons émis l’hypothèse que l’activation immunitaire chronique plus élevée rapportée chez des individus co-infectés par le VIH-1 et VHC pourrait être due à un dysfonctionnement de la voie de TLR7/IFN-I dans les pDCs. Nos données montrent que le VHC entraîne, chez des individus infectés par le VIH-1, des altérations associées aux pDCs et à l’IFNa, qui sont associées ˆ la sévérité de la maladie hépatique. Nos résultats suggèrent que les patients co-infectés par le VIH-1 et le VHC, même à fibrose minime, pourraient bénéficier d’un traitement plus précoce. / Type I interferons (IFN) can be produce by any cell type but plasmacytoid dendritic cells (pDC) are the main producers. IFNa orchestrates multiple pathogenic mechanisms in human immunodeficiency virus 1 (HIV-1). Studying physiological and pathophysiological model scan provide critical informations on how to harness IFNa signaling for therapeutic purposes. pDCs from females produce more IFNa upon Toll-like receptor (TLR) 7 stimulation than pDCs from males. The mechanisms underlying such difference have only been partially identified. We demonstrate here a mechanism by which increased IRF5 expression in females, under the control of the esrogen receptor a, contribute to increased IFN? production upon TLR7 stimulation. HCV co-infection is one of the major cause of mortality among HIV-1 infected individuals. We hypothesized that increased chronic immune activation observed in HCV-HIV-1 co-infected individuals may be related to altered TLR7/IFNa signaling in pDCs. Our data show that HCV triggers alterations in pDCs and IFNa signaling in HIV-1 co-infected individuals, which are associated to hepatic disease severity. Our results suggest that HCV-HIV-1 co-infected individuals, even with minimal fibrosis, may benefit from ealier treatment initiation.

Human Immunodeficiency Virus (HIV) is a virus that is transmitted through certain bodily fluids and compromises the immune system of its host. Despite the emergence of antiretroviral therapy (ART) converting human immunodeficiency virus type 1 (HIV-1) infection from a fatal disease to a chronic condition, there is still no cure. ART frequently reestablishes peripheral CD4+ T cell counts, but persistent immune dysfunction and inflammation strongly correlate with increased risks of attaining non-AIDS morbidity and mortality. Elimination of this reservoir may occur by the proposed mechanism of combining latency-reversing agents (LRAs) with immune effectors, such as CD8+ T cells (Meås et al., 2020). Here, our study investigates Toll-like receptor 7/8 (TLR 7/8) superagonists that may act as potent, effective latency reversal agents (LRAs). Whether this will prove to be the case needs to be further studied, and potential adverse toxicities must be identified. Whether comparable results will be observed in peripheral blood mononuclear cells (PBMCs) infected with HIV-1 as in our study using PBMCs infected with simian immunodeficiency virus (SIV) remains to be tested. Our results provide further hope for a potential cure for HIV-infected individuals.

Les récepteurs Toll-like 7 et 8 jouent un rôle important dans l’activation de la réponse immunitaire innée et adaptative. Leur stimulation conduit à la production des cytokines pro-inflammatoires et d’interférons de type I. L’imiquimod et son dérivé le résiquimod sont les premières molécules de faible poids moléculaire décrites comme agonistes du TLR7 et TLR8. Ces deux molécules ont montré des activités anticancéreuses et adjuvantes très importantes. Récemment, les TLR 7 et 8 ont fait l’objet de plusieurs publications visant à développer de nouveaux agonistes TLR7 et/ou TLR8 dans la perspective d’être utilisés comme adjuvants vaccinaux. Malgré les rôles essentiels de TLR7 et TLR8 dans la stimulation du système immunitaire, une activation immunitaire chronique peut être responsable de plusieurs maladies infectieuses et auto-immunes. D’où l’importance de développer également des antagonistes TLR7 et/ou TLR8.Ce travail de thèse est consacré à la synthèse et le développement de nouvelles molécules hétérocycliques, analogues de l’imiquimod et de résiquimod, dans le but d’identifier de nouveaux ligands TLR7 et/ou TLR8. Des voies de synthèse innovantes, permettant une modulation chimique importante grâce à des couplages croisés pallado-catalysés, ont été mises au point et ont permis d’obtenir une cinquantaine de molécules appartenant à trois séries chimiques différentes de type imidazo[1,2-a]pyrazine, imidazo[1,5-a]quinoxaline et pyrazolo[1,5-a]quinoxaline. De nombreux essais d’alkylation ont été tentés sur ces trois séries chimiques afin d’introduire une large variété de substituants sur le cycle à cinq sommets. L’application du couplage croisé de Sonogashira nous a permis d’établir une liaison C-C et introduire diverses chaines alkyles. Ces composés ont été testés pour leur activité agoniste et antagoniste TLR7 et 8. Aucun des composés cibles n'a présenté d’activité agoniste TLR7 et TLR8, dans l'intervalle des concentrations testées. Par contre, tous les composés ont montré une activité antagoniste sélective du TLR7. Les composés les plus actifs, 5.35a et 5.35b, membres de la série pyrazolo[1,5-a]quinoxaline ont montré des IC50 de l’ordre de 10 μM. Ces résultats prometteurs nous ont permis la découverte d’une activité antagoniste TLR7 importante pour la série pyrazolo[1,5-a]quinoxaline, une série très peu développée dans la littérature. La modulation chimique des molécules actives nous permet de donner naissance à de nouveaux leaders, qui peuvent jouer un rôle important dans la thérapie de plusieurs maladies infectieuses et auto-immunes. / Toll-like receptors 7 and 8 play an important role in immune system activation. Their stimulation leads to the production of pro-inflammatory cytokines and type I interferons. Both receptors recognize viral ssRNA, as well as synthetic tricyclic imidazoquinoline derivatives such as imiquimod (TLR7 agonist) and resiquimod (TLR7/8 agonist). These two molecules showed significative anti-cancer and adjuvant activities. Many reports in the literature have been focused on the development of new TLR7/8 agonists belonging to different chemical series. These agonists strongly induce the production of T helper 1-polarizing cytokines and may therefore serve as promising candidate vaccine adjuvants. Despite the essential roles of TLR7 and TLR8 in the immune system stimulation, chronic immune activation may be responsible for several infectious and autoimmune diseases. Consequently, the development of TLR7 inhibitors may play an important role in the therapy of these diseases.In this study, we are interested in the synthesis and development of new heterocyclic molecules, analogs of imiquimod and resiquimod, in order to identify new TLR7 and/or TLR8 ligands. Different synthetic pathways have been developed, using cross coupling reactions, in order to obtain a wide variety of molecules belonging to three chemical series: imidazo[1,2-a]pyrazine, imidazo[1,5-a]quinoxaline et pyrazolo[1,5-a]quinoxaline. Various alkylation reactions were attempted on these three chemical series in order to introduce a wide variety of substituents on the five-membered ring. The application of Sonogashira's cross-coupling allowed us to establish a C-C bond and introduce various alkyl chains. All compounds have been tested for their TLR7/8 agonistic and antagonistic activity using HEK-Blue™-hTLR7/8 cells. The synthesized compounds are completely inactive as TLR7/8 agonists and are selective TLR7 antagonists. Two compounds of the pyrazolo[1,5-a]quinoxaline series, compound 5.35a and 5.35b, bearing butyl and isobutyl chain respectively, are potent and selective TLR7 antagonists with low micromolar IC50. Results allowed us to discover significative activity for the pyrazolo[1,5-a]quinoxaline series as selective TLR7 antagonists, which may therefore play an important role in the therapy of several infectious or autoimmune diseases.

Hepatitis C Virus (HCV) is the primary cause of liver transplantation due to its chronic nature in up to eighty percent of infected cases. Around 3 percent of the world’s population is infected with HCV. Treatment for HCV is a combined Ribavirin and interferon-α (IFN-α) therapy effective in only fifty to eighty percent of patients depending on HCV genotype. The growing health concern with this disease is the lack of a cure despite liver transplantation. HCV targets hepatocytes, liver cells, but is not cytolytic. HCV has been shown to induce end stage liver disease through sustained inflammation from the host’s immune system in the liver. One of the key dilemmas in HCV research and the search for fully effective treatments or vaccines is the lack of animal models. HCV infectivity and disease is limited to primates, most specifically to humans, which cannot be fully replicated in any other living being. The mechanisms for HCV evasion or activation of the immune system are complex, many and discoveries within this field are crucial to overcoming this destructive hepatic infection. Toll-like receptors (TLR) are cellular activators of the innate immune system that have been a target of HCV. Activated TLRs trigger both the inflammatory and anti-viral pathways to produce inflammatory cytokines and interferons. HCV proteins have been reported to activate a number of TLRs in a variety of cell types. In order to identify possible targets of HCV within the TLR family, we first characterized TLR presence and function in both human hepatic carcinoma cell lines and purified primary human hepatocytes. RNA from TLRs 1-10 was observed to varying degrees in both the hepatoma cell lines and the primary hepatocytes. We show the extracellular and/or intracellular presence of TLR2, TLR1, TLR3 and TLR7 proteins in hepatoma cell lines. TLR3 and TLR7 are located within the endosome and recognize viral RNA products. We recently reported that TLR2-mediated innate immune signaling pathways are activated by HCV core and NS3 proteins. TLR2 activation requires homo- or heterodimerization with either TLR1 or TLR6. We show NF-κB activation in hepatoma cells by TLR2/1, TLR2/6 ligand and HCV protein stimulation. In primary hepatocytes, HCV proteins induced both IL-8 and IL-6 production. We also show that primary hepatocytes initiate a Type 1 IFN response in addition to IL-8 and IL-6 production upon stimulation with a TLR7/8 ligand. Human hepatoma and primary hepatocytes are responsive to TLR2, TLR1, TLR6, TLR7/8 ligands and HCV proteins. Activation of these TLRs may contribute to the inflammatory mediated destruction caused by HCV or could be targets of HCV contributing to its immune evasion. We found previously that hepatoma cells and primary hepatocytes are responsive to TLR2 ligands and HCV proteins. We also reported that TLR2 is activated by HCV proteins. Here we aimed to determine whether TLR2 coreceptors participated in cellular activation by HCV core or NS3 proteins. By designing siRNAs targeted to TLR2, TLR1 and TLR6, we showed that knockdown of each of these receptors impairs pro- and anti-inflammatory cytokine activation by TLR-specific ligands as well as by HCV core and NS3 proteins in Human Embryonic Kidney cells (HEK/TLR2) and in primary human macrophages. We found that HCV core and NS3 proteins induced TNF-α and IL-10 production in human monocyte-derived macrophages, which was impaired by TLR2, TLR1 and TLR6 knockdown. Contrary to human data, results from TLR2, TLR1 or TLR6 knockout mice indicated that the absence of TLR2 and its coreceptor TLR6, but not TLR1, prevented the HCV core and NS3 protein-induced peritoneal macrophage activation. TLR2 may utilize both TLR1 and TLR6 coreceptors for HCV core- and NS3-mediated activation of macrophages and innate immunity in humans. These results imply that multiple pattern recognition receptors could participate in cellular activation by HCV proteins contributing to inflammatory disease. Two critical factors in chronic HCV infection are inflammatory disease and immune evasion. We have demonstrated that TLR2 and its co-receptors play a role in inflammatory-mediated induction via HCV NS3 and core administration. It has recently been shown that HCV targets the TLR3 pathway to aid in immune evasion. TLR3 is only one of four viral recognition receptors located within the endosome and it is plausible that HCV may target others. We hypothesized that HCV infection may interfere with the expression and function of TLR7, a sensor of single stranded RNA. Investigating any effect on TLR7 by HCV may reveal a new mechanism for HCV immune evasion. Low levels of both TLR7 mRNA and protein were measured in HCV replicating cells compared to control cells while reducing HCV infection with either IFNα or restrictive culture conditions restored the decreased TLR7 expression. Downstream of the TLR7 pathway, an increased baseline IRF7 nuclear translocation was observed in HCV replicating cells compared to controls. Stimulation with a TLR7 ligand, R837, resulted in significant IRF7 nuclear translocation in control cells. In contrast, HCV replicating cells showed impaired IRF7 activation. Use of RNA polymerase inhibitors on hepatoma cells, control and HCV replicating, revealed a shorter TLR7 half life in HCV replicating cells compared to control cells which was not seen in TLR5 mRNA. These data suggest that reduced TLR7 expression, due to RNA instability, directly correlates with HCV replication and results in impaired TLR7-induced IRF7-mediated cell activation. In conclusion, Hepatitis C Virus manipulates specific Toll-like receptors’ expression and their signaling pathways to induce cytokine production. HCV utilizes surface receptors TLR2 and its co-receptors which once activated could contribute to inflammatory disease by production of inflammatory cytokines and possibly immune evasion. HCV down-regulates TLR7, a viral recognition receptor, by decreasing mRNA stability which could facilitate evasion of host immune surveillance.

Submitted by Nadir Basilio (nadirsb@uninove.br) on 2016-05-25T16:04:47Z No. of bitstreams: 1 Nicole Cristine Rigonato de Oliveira.pdf: 1240943 bytes, checksum: 14fa67b6692555f3bc76fb1f34186220 (MD5) / Made available in DSpace on 2016-05-25T16:04:47Z (GMT). No. of bitstreams: 1 Nicole Cristine Rigonato de Oliveira.pdf: 1240943 bytes, checksum: 14fa67b6692555f3bc76fb1f34186220 (MD5) Previous issue date: 2014-12-19 / The acute respiratory distress syndrome (ARDS) is a disease characterized by respiratory failure due to an inflammatory response, which has high morbidity and mortality. Several cytokines seem to orchestrate the acute and chronic processes, mainly mediated by toll like receptors (TLRs). The literature demonstrates that aerobic exercise (AE) is capable of decreasing the secretion of pro-inflammatory cytokines in the lungs mediated increased release of interleukin 10 (IL-10), and AE modulate expression of TLRs and antioxidant enzymes. The objective of this study was to evaluate whether the anti-inflammatory effects of AE in an experimental model of intra and extrapulmonary ARDS are mediated by IL-10. For this, the animals were subjected to physical training on a treadmill, moderate for 4 weeks. 24 hours after the last physical test, animals received LPS by intratracheally (it) (10ug / animal) or intraperitoneally (ip) (100ug / animal). After 24 hours, the animals were assessed for the number of cells and pro- and anti-inflammatory cytokines in bronchoalveolar lavage fluid (BAL) and serum were the number of neutrophils in the lung parenchyma and expression of TLR4, TLR7, SOD, ânion and QL in lung homogenates. AE reduced accumulation of neutrophils in the lung parenchyma, so it LPS and LPS ip (p <0.01) and BAL (p <0.01). AE attenuate the levels of proinflammatory cytokines in BAL and serum (p <0.05), as measured by ELISA. AE had levels of anti-inflammatory cytokine IL-10 increased in the serum and BAL (p <0.05). AE also reduced the expression of TLR4 in lung homogenates (p <0.05) and increased the expression of TLR7 in group LPS + AE evaluated by western blotting (p <0.05). The results for the antioxidant enzyme SOD showed a significant increase in the groups submitted to AE. It follows that the impact of reduced LPS-induced ARDS, regardless of etiology, and these effects appear to be mediated by modulation of the AE secretion of anti-inflammatory cytokines, especially IL-10, modulating TLR4 and TLR7 and increased expression of SOD (p <0.05). / A síndrome do desconforto respiratório agudo (SDRA) é uma doença caracterizada pela insuficiência respiratória decorrente a uma resposta inflamatória, que apresenta alta morbi-mortalidade. Diversas citocinas parecem orquestrar os processos agudo e crônico, mediados principalmente por receptores toll like (TLRs). A literatura demonstra que o exercício aeróbio (EA) é capaz de diminuir a secreção de citocinas pró-inflamatórias nos pulmões, mediado pelo aumento da liberação de interleucina 10 (IL-10), além de o EA modular a expressão de TLRs e enzimas antioxidantes. Assim, o objetivo desse estudo foi avaliar se os efeitos anti-inflamatórios do EA em modelo experimental da SDRA intra e extrapulmonar são mediados por IL-10. Para isso, os animais foram submetidos ao treinamento físico em esteira, de intensidade moderada, durante 4 semanas. Após 24 horas ao último teste físico, os animais receberam LPS por via intra-traqueal (it) (10ug/animal) ou por via intra-peritoneal (ip) (100ug/animal). Após 24 horas, os animais foram avaliados para o número de células e de citocinas pró e anti-inflamatórias no fluído do lavado broncoalveolar (LBA) e no soro, número de neutrófilos no parênquima pulmonar e expressão do TLR4, TLR7, SOD, ânion e QL nos homogenatos de pulmão. EA reduziu a acumulação de neutrófilos no parênquima pulmonar, tanto LPS it e LPS ip (p <0,01) e no LBA (p <0,01). EA atenuou os níveis de citocinas pró-inflamatórias no LBA e soro (p <0,05), avaliada por ELISA. EA teve os níveis da citocina anti-inflamatória IL-10 aumentados no soro e LBA (p <0,05). EA também reduziu a expressão de TLR4 nos homogenatos de pulmão (p <0,05) e aumentou a expressão de TLR7 no grupo de LPS + EA avaliada por western blotting (p <0,05). Os resultados para a enzima antioxidante SOD apresentou aumento significante nos grupos submetidos ao EA. Conclui-se que EA reduziu o impacto de SDRA induzida por LPS, independente da etiologia e tais efeitos parecem estar mediados pela modulação do EA na secreção de citocinas anti-inflamatórias, principalmente da IL-10, na modulação de TLR4 e TLR7 e no aumento da expressão de SOD (p<0,05).

Albert Font Haro ABSTRACT Modulation of plasmacytoid dendritic cell function: role of immunoreceptors TIM-3 and BDCA-2 Plasmacytoid dendritic cells (pDCs) are key players in the antiviral response as well as in linking innate and adaptive immune response. They express endosomal toll-like receptors 7 and 9, which can detect ssRNA and unmethylated CpG DNA, respectively. Due to the constitutive expression of the transcription factor IRF7, pDCs are able to rapidly produce massive quantities of type I (α, β, ω) and type III (1, 2, 3, 4) interferons (IFN-I and IFN-III) as well as pro- inflammatory cytokines such as IL-1, IL-6 and TNF-α. After maturation, they also function as antigen-presenting cells. Despite intense research, the mechanisms of IFN and pro-inflammatory cytokines production and regulation are still poorly understood. Using the pDC cell line GEN2.2 and also primary human pDCs, we shed light on the role of kinases MEK and SYK in IFN-I production and regulation. We found that SYK is not only involved in the regulatory receptor (RR)-mediated BCR-like pathway that represents the negative regulation of IFN-I and IFN-III secretion but also in the positive TLR7/9-mediated signal transduction pathway that leads to IFN-I production, representing the immunogenic function. We also found that MEK plays a...