Au cours de ce travail, nous nous sommes intéressés à développer des voies de synthèse convergentes et diastéréosélectives en vue de préparer des analogues pyrrolidiniques des alcaloïdes de Lobelia comme nouveaux ligands des récepteurs nicotiniques centraux à l’acétylcholine. Ainsi, nous avons mis au point une méthode « bidirectionnelle » basée sur des réactions de double aza-Michael et donnant accès à des pyrrolidines 2,5-disubstituées. Une étude de réactivité a également été mené afin d’améliorer la chimiosélectivité des différents processus réactionnels impliquant des réactions d’aza-Michael dans des séquences de cyclisation tandem. Dans un second temps, nous avons décrit une voie dite « d’élongation monodirectionnelle » permettant d’accéder à des 2,5-trans-pyrrolidines énantiopures. Enfin, la dernière partie de ce manuscrit aborde une étude prospective de réductions désymétrisantes pour la synthèse d’homologues pyrrolidiniques de la lobéline. / The present work has been dedicated to the development of convergent and diastereoselective routes for the preparation of pyrrolidine Lobelia alkaloid analogues as novel neuronal nicotinic receptors. We have settled a selective bidirectional strategy based on chain homologation by double olefination followed by aza-Michael reactions as a straightforward access to 2,5-cis-disubstituted pyrrolidines that was extended to the synthesis of 2,6-cis-piperidines. Additional studies have been carried out in order to drive chemoselectivities in the course of competitive tandem aza-Michael-induced ring closure reactions. In the same time, we also described a monodirectional route to access 2,5-trans-disubstituted pyrrolidines. The last part of this manuscript has been finally dedicated to a prospective reductive desymmetrization study for the rapid and enantioselective synthesis of pyrrolobeline homologues.

A nicotina é um alcalóide extraído da planta do tabaco e age como agonista em neurônios e outras células que expressam receptores nicotínicos. Essa substância é capaz de atravessar a barreira hematoencefálica e mimetizar os efeitos da acetilcolina endógena ao interagir com os diferentes subtipos de receptores. O hipocampo e o cerebelo são regiões encefálicas ricas em receptores nicotínicos como o alfa7 e o alfa42 que estão também envolvidos na progressão de doenças neurodegenerativas, como a doença de Alzheimer, e neuropsiquiátricas, como a esquizofrenia, ambas apresentando um contexto inflamatório. O lipopolissacarídeo (LPS) é um indutor inflamatório que age via receptor toll-4 (TLR-4) ativando, dentre outros, o fator de transcrição NF-capaB. Vários estudos têm demonstrado tanto in vitro quanto in vivo que a exposição crônica à nicotina inibe a resposta imune. Uma vez que o fator de transcrição NF-capaB regula a expressão gênica de diversos fatores que medeiam a plasticidade e o desenvolvimento neuronal e inflamação, nosso objetivo foi avaliar os efeitos da nicotina crônica sobre a via de sinalização induzida pelo LPS. Sendo assim, avaliou-se a translocação nuclear do NF-capaB, a expressão de indutores inflamatórios (TNF, IL-1 e iNOS) e também do receptor TLR-4 tanto no hipocampo quanto no cerebelo. Além disso, foram determinados os níveis plasmáticos das citocinas TNF, IL-1, MIP-1alfa, CINC2alfa/. Nossos resultados mostram que, perifericamente, o LPS induziu a produção das citocinas plasmáticas, mas a nicotina não foi capaz de reverter seus efeitos. Já no cerebelo e no hipocampo, a nicotina crônica (0,1 e 1,0 mg/kg) interferiu significativamente na translocação do NF-capaB induzida pelo LPS, levando inclusive a um bloqueio importante da expressão do mRNA de TNF, IL-1, iNOS no hipocampo, e de IL-1 no cerebelo. Ademais, no hipocampo, a nicotina também interferiu na expressão do receptor TLR-4. A mecamilamina, antagonista heteromérico não-seletivo não foi capaz de reverter os efeitos da nicotina sobre o NF-capaB no hipocampo, mas o fez no cerebelo. Já o antagonista seletivo de receptor alfa 7, metililaconitina, bloqueou a ação da nicotina no hipocampo. / Nicotine is an alkaloid extracted from Tobacco plants, and it acts as an agonist in neurons and other cells that express nicotinic receptors. This substance crosses the blood brain barrier and mimics endogenous acetylcholine in interacting with various receptor subtypes. The hippocampus and cerebellum are brain regions rich in nicotinic receptors including 7, 42 and other subtypes, involved in neurodegenerative and neuropsychiatric disorders, including Alzheimer disease and schizophrenia, respectively, in a neuroinflammatory context. The lipopolysaccharide (LPS) is a known inflammatory inducer that exerts its effects through the Toll-like receptor 4 (TLR-4) and activates the transcription factor NF-capaB. In the present work, we evaluate the effects of chronic nicotine on the nuclear translocation of NF-capaB, on the expression of NF-capaB regulated inflammatory inducers (TNF, IL-1 and iNOS) and on the LPS-induced TLR4 mRNA expression in response to chronic nicotine treatment in hippocampus and cerebellum. In addition, we accessed the production of peripheral cytokines (TNF, IL-1, MIP-1, CINC 2/). Our results demonstrate that LPS induced the production of peripheral cytokines but nicotine treatment did not interfere in their plasma levels. However, in cerebellum and hippocampus, chronic nicotine 1.0 and 0.1 mg/kg interfered in the LPS-induced NF-capaB leading to a deficient mRNA expression of its related genes TNF, IL-1 and iNOS in hippocampus and of IL-1, in cerebellum. Chronic nicotine (0.1 mg/kg, s.c.) significantly interfered in the TLR4 mRNA expression in LPS-challenged animal group in hippocampus. The non-selective heteromeric antagonist mecamylamine (1.0 mg/kg s.c.) reverted the nicotine effects over NF-capaB nuclear translocation in cerebellum, but not in hippocampus. On the other hand, the 7 antagonist methyllycaconitine (MLA) (5.0 mg/kg i.p.) reverted the nicotine effects over NF-capaB in hippocampus but not in cerebellum. Nicotine prevents LPS effects, both in cerebellum and hippocampus, once it reduced significantly the nuclear translocation of NF-capaB and the expression of related pro-inflammatory cytokines. Besides, the nicotinic effects seem to be mainly mediated by heteromeric receptors in cerebellum and through 7 receptors in hippocampus.

Os receptores nicotínicos de acetilcolina (colinérgicos) (nAChRs) neuronais são proteínas integrais de membrana e pertencem à família de canais iônicos controlados por ligante, compostos por subunidades α e β. Esses receptores desempenham um papel-chave na transmissão de sinal entre os neurônios nos sistemas nervoso central e periférico. O subtipo α3β4, por exemplo, é o nAChR neuronal mais expresso no sistema nervoso autônomo; nAChRs contendo a subunidade α3 estão presentes em alta densidade no gânglio cervical superior, glândulas pineal e adrenais. Também estão presentes na substancia nigra, striatum, hipocampo, locus ceruleus, tracto habênulo-interpeduncular e cerebelo. Os nAChRs são inibidos por uma variedade de substâncias químicas, incluindo toxinas naturais, anestésicos locais, drogas de abuso (por, exemplo, cocaína) e compostos clinicamente importantes (tranqüilizantes, por exemplo). O mecanismo de inibição desses receptores tem sido investigado intensivamente. Neste estudo, nós investigamos o mecanismo pelo qual a tacrina (9-1,2,3,4-tetraidroaminoacridina), um agente usado clinicamente no tratamento da doença de Alzheimer, inibe o nAChR α3β4 de rato recombinante expresso nas células KXα3β4R2, utilizando uma técnica de cinética química rápida. A constante de dissociação da nicotina do sítio que controla a ativação do receptor, Kd, é 23 µM e a constante de equilíbrio de abertura do canal, Φ-1, é 4. A tacrina inibe o receptor competitivamente, com um KI de 0,77 µM. / Neuronal nicotinic acetylcholine (cholinergic) receptors (nAChRs) are integral membrane proteins and belong to the family of ligand-gated cation channels composed by α and β subunits. These receptors play a key role in the signal transmission between neurons in the central and peripheral nervous system. The α3β4 subtype, for example, is the most expressed neuronal nAChR in autonomic ganglia; α3-containing nAChRs are present at particularly high density in the superior cervical ganglia, pineal, and adrenal glands. They are also present in the substancia nigra, striatum, hippocampus, locus ceruleus, habenulo-interpeduncular tract and cerebellum. The nAChRs are inhibited by a variety of chemical substances, including natural toxins, local anesthetics, abused drugs (e.g., cocaine) and clinically important compounds (e.g., tranquilizers). The mechanism of inhibition of these receptors has been intensively investigated. In this study, we investigated the mechanism by which tacrine (9-1,2,3,4-tetahydroaminoacridine), an agent used clinically to treat Alzheimers disease, inhibits the recombinant rat α3β4 nAChR expressed in KXα3β4R2 cells, using a rapid chemical kinetic technique. The nicotine dissociation constant for the site controlling receptor activation, Kd, is 23 µM and the channel-opening equilibrium constant, Φ-1, is 4. Tacrine inhibits the receptor competitively, with a KI of 0.77 µM.

Parmi les facteurs de risque environnementaux de l'arthrose, la consommation de tabac occupe une place importante, mais reste encore controversée. Parmi les 4000 composés présents dans la cigarette, la nicotine est l'une des molécules les plus actives physiologiquement. Au cours de ce travail, nous avons étudié l'impact de la nicotine sur les chondrocytes humains et la prolifération et la différenciation chondrogénique des cellules souches mésenchymateuses de la gelée de Wharton (CSM-GW). Nous avons trouvé que la nicotine aux concentrations utilisées n’a pas d’effet sur la prolifération cellulaire, mais induit une augmentation de l’expression de la métalloproteinase matricielle (MMP13) dans les chondrocytes humains. Ces données suggèrent que la nicotine a un effet pro-catabolique sur les chondrocytes humains, en stimulant la dégradation des composants matriciels. Chez des patients arthrosiques fumeurs, les voies de synthèse et de dégradation des composants matriciels sont plus activées dans les chondrocytes par rapport aux non fumeurs. De plus, la nicotine inhibe la prolifération et la migration cellulaire de CSM-GW, et présente un effet délétère sur la chondrogénèse de CSM-GW. Elle stimule la réaction inflammatoire et la différenciation hypertrophique. Nous avons montré, pour la première fois, l'expression du nicotinic acetylcholine receptor (nAChR), en particulier de la sous unité α7 dans les CSM-GW aux niveaux transcriptionnel et traductionnel. L’effet délétère de la nicotine sur les CSM-GW serait probablement médiée par la sous unité α7 nAChR, De façon intéressante, l’α-Bungarotoxin (α-BTX), inhibiteur spécifique de α7 nAChR, peut réverser cet effet partiellement. En conclusion, nous suggérons que la nicotine pourrait altérer l’ontogenèse du cartilage, et induire potentiellement l’augmentation de la prévalence de l’arthrose chez l’adulte / Among the environmental risk factors for osteoarthritis (OA), tobacco consumption features prominently but is still controversial today. Among the 4,000 compounds present in cigarette smoke, nicotine is one of the most physiologically active molecules. The aim of the study is to measure the impact of nicotine on human chondrocytes and on the proliferation and chondrogenic differentiation of Wharton’s Jelly stem cells (WJ-MSC). We found that nicotine at the concentrations used had no effect on the proliferation of primary human OA chondrocytes, but induced an increase in the expression of matrix metalloproteases (MMP13). It unravels the catabolic effects of nicotine in the joint by stimulating matrix degradation. This result suggests a pro-catabolic effect of nicotine in the joint by stimulating matrix degradation. In smokers, the synthesis as well as the degradation pathways in chondrocytes are stimulated, when compared to no smokers. In addition, the cell proliferation and migration of WJ-MSC were significantly impaired by nicotine, and it also had an adverse effect on the chondrogenesis of WJ-MSC by stimulating the inflammatory response and hypertrophic differentiation. We have shown, for the first time, the expression of the nicotinic acetylcholine receptor (nAChR), in particular that of the α7 subunit in the WJ-MSC at the transcriptional and translational levels. The adverse effect of nicotine on WJ-MSC was probably mediated by α7 nAChR. Interestingly, α-Bungarotoxin (α-BTX), a specific inhibitor of α7 nAChR, could partially reverse this effect. In conclusion the results show that nicotine has an adverse effect on the ontogenesis of cartilage, and potentially induces an increase in the prevalence of osteoarthritis in adults

Introdução: Colite Ulcerativa (UC) é uma Doença Inflamatória Intestinal (DII) caracterizada por uma resposta imune exacerbada, com sintomas como diarreia, perda de peso e sangue nas fezes. Apesar dos medicamentos disponíveis, a remissão da doença nem sempre consegue ser alcançada e há a necessidade de terapias alternativas. A colite induzida por DSS (Dextran Sulfate Sodium) é um modelo animal utilizado na investigação de novas terapias por sua semelhança à UC humana. DSS provoca dano à barreira epitelial do cólon, induzindo uma resposta imune exacerbada; entretanto, o exato mecanismo não está totalmente esclarecido. O Ultrassom Terapêutico (TUS) foi utilizado para tratamento de injúria renal em modelo experimental, sua ação se dá através da estimulação do nervo vago (VN) e consequente ativação da via antiinflamatória colinérgica (CAIP). Uma vez que pacientes com DII podem exibir atividade disfuncional do VN, TUS pode ser investigado como terapia alternativa. Objetivos: Investigar temporalmente o perfil clínico, proteômico, histológico e imunológico da colite aguda induzida por DSS; e determinar os efeitos de TUS na colite induzida por DSS. Métodos: No primeiro estudo, a severidade da colite foi avaliada pela administração de DSS 1-3%, observando a resposta clínica e histológica. A análise temporal de DSS 3% incluiu uma avaliação proteômica e histológica do cólon, e a resposta imune celular no baço, linfonodo mesentérico (MLN) e cólon. No segundo estudo, utilizando o modelo de DSS 2%, TUS foi aplicado no abdômen dos animais e foram observados os sintomas clínicos, dano histológico, proteômica do cólon e respostas imunes celulares no baço, MLN e cólon. Animais esplenectomizados ou knockout para a7nAChR (marcador clássico para ativação de CAIP) foram utilizados. Resultados: No primeiro estudo, observou-se que a severidade da doença foi aumentada seguindo concentrações de 1-3% DSS. A análise temporal de DSS 3% demonstrou que os macrófagos (F4/80+) se apresentam como a primeira resposta celular, seguidos por células T CD25+, CD4+ e CD8+. A piora clínica da doença correspondeu ao aumento progressivo de fatores pró-inflamatórios e dano tecidual no cólon, exceto no dia 8. Foram observados menores níveis dos marcadores de células T CD25+, CD4+ e CD8+ no MLN e/ou baço, sugerindo a ocorrência de tropismo destas células para o intestino. No segundo estudo, a aplicação de TUS diminuiu a severidade da doença através da melhora de sintomas clínicos, danos teciduais e encurtamento do cólon. A proteômica do cólon demonstrou uma resposta anti-inflamatória durante a fase de injúria (D0-7), induzindo uma resolução acelerada da doença na fase de recuperação (D8-14). TUS diminuiu os níveis de células T CD8+ e normalizou os níveis de células T CD25+ no cólon. Animais esplenectomizados não demonstraram melhora clínica ou histológica, enquanto animais a7nAChR KO apresentaram piora da colite experimental. Além disso, TUS aumentou os níveis de células F4/80+a7nAChR+ no intestino de animais WT DSS 2%. Conclusão: Nossos resultados demonstram que a severidade da doença depende da concentração de DSS, relacionada com as respostas clínica, proteômica e imune no modelo animal de DSS 3%; e TUS diminuiu a severidade da colite induzida por DSS presumidamente pela da estimulação do VN e consequente ativação de CAIP através do baço. / Introduction: Ulcerative Colitis (UC) is an Inflammatory Bowel Disease (IBD) characterized by uncontrolled immune response, presenting with symptoms of diarrhea, weight loss and bloody stools. Despite available treatments, UC sustained remission is not achievable and there is still the need for alternative therapies. Dextran Sulfate Sodium (DSS)-induced colitis is a mouse model used to investigate novel therapies, since it closely mimics human UC. DSS damages the colonic epithelial barrier, leading to an exacerbated immune response. However, the exact mechanism is not totally understood. Previous studies showed the use of Therapeutic Ultrasound (TUS) to prevent kidney injury in mice through stimulation of the vagus nerve (VN) and activation of the cholinergic anti-inflammatory pathway (CAIP). Since IBD patients can present with dysfunctional VN activity, TUS could be studied as an alternative therapy. Objectives: To investigate the temporal clinical, proteomic, histological and cellular immune profiles of DSS-induced acute colitis; and to determine the effects of TUS directed toward the VN and spleen in the course of DSS-induced colitis. Methods: First, we analyzed DSS-induced colitis severity by administration of 1-3% DSS, observing the clinical course and histological damage. A time course analysis was performed at 3% DSS, including colon proteomics, colon histology and immune cell responses in the spleen, MLN (mesenteric lymph node) and colon. Next, utilizing 2% DSS in drinking water, we applied TUS over the mice abdomen and analyzed clinical symptoms, histological damage, colon proteomics and immune cell responses in the spleen, MLN and colon. Splenectomized and a7nAChR (key indicator of CAIP activation) KO animals were also used. Results: In the first study, we observed worsening of the disease when increasing DSS concentrations from 1 to 3%. Time course analysis of 3% DSS revealed macrophages to be the first responders, followed by CD25+, CD4+ and CD8+ T cells. Worsening of the disease corresponded to a progressive increase in pro-inflammatory colonic factors and histological damage, except at day 8. Lower levels of CD25+, CD4+ and CD8+ T cells in MLN and/or spleen suggest an immune cell tropism to the gut. In the second study, TUS attenuated DSS induced colitis through amelioration of clinical symptoms, histological damage and colon shortening. Proteomic colon analysis demonstrated an antiinflammatory profile during the injury phase (D0-7), whilst inducing an early resolution of the disease during the recovery phase (D8-14). TUS decreased CD8+ and normalized CD25+ T cell levels in the gut. Splenectomized animals demonstrated no improved clinical and pathological outcomes, and a7nAChR KO mice presented with worsening of the disease. Furthermore, there were increased levels of F4/80+a7nAChR+ cells in the colon of 2% DSS WT mice under TUS treatment. Conclusion: Our results demonstrate that the severity of colitis is dependent on DSS concentration, correlated with clinical, proteomic and cellular immune responses on 3% DSS; and TUS significantly improved DSS-induced acute colitis presumably through stimulation of the VN and consequent activation of CAIP through the spleen.

Introdução: A inflamação constitui um dos fatores mais importantes da fisiopatologia da asma brônquica, caracterizada por uma resposta eosinofílica com produção de citocinas de perfil Th2. A persistência desta inflamação induz no pulmão um processo de reparo associado à redução progressiva da função pulmonar, que nem sempre é revertida pelos tratamentos disponíveis. O sistema colinérgico anti-inflamatório é descrito como um mecanismo neural que suprime a resposta imune e controla a inflamação principalmente pelo efeito da acetilcolina em receptores nicotínicos do tipo alfa7 (alfa7nAChR) encontrados em células do sistema imune. A acetilcolina (ACh) exerce um importante efeito na asma e recentemente demonstramos que a redução parcial da liberação da acetilcolina induz per se a inflamação pulmonar. Embora se saiba que os receptores muscarínicos (mAChRs) exercem um efeito pró-inflamatório e broncoconstritor na asma, a ativação de receptores nicotínicos (nAChRs) poderia ter um efeito benéfico reduzindo a inflamação pulmonar, fato demonstrado em modelos de inflamação sistêmica e aguda. O efeito da ativação do alfa7nAChR na fisiopatologia da asma ainda não está claramente elucidado. Objetivo: Investigar o efeito do tratamento com PNU-282987 (PNU), um agonista específico do alfa7nAChR, em um modelo murino de inflamação alérgica crônica das vias aéreas. Metodologia: Camundongos BALB/c foram submetidos ao protocolo de indução alérgica crônica das vias aéreas com ovoalbumina (OVA) ou salina intraperitoneal (i.p.) e posterior desafios inalatórios. A partir do 22° dia, os animais receberam diariamente tratamento com PNU ou veículo (Ve) até o 28° dia. Foram testadas três doses de PNU (0,5, 1,0 e 2,0 mg/Kg). A fim de evidenciar se o efeito obtido no tratamento com PNU era dependente do receptor alfa7nAChR, um grupo de animais foi tratado com MLA (antagonista do alfa7nAChR), previamente ao tratamento com PNU. No 29° dia do protocolo, os animais foram eutanasiados e foram avaliados o número de células inflamatórias no lavado broncoalveolar (LBA) e no sangue, os níveis de citocinas no LBA, a expressão do alfa7nAChR e mAchRs do tipo 3 (M3) e a ativação do fator de transcrição nuclear kB (NF-kB) no pulmão. O remodelamento brônquico foi avaliado por morfometria. As análises estatísticas foram realizadas por meio do programa SigmaStat (Jandel Scientific, San Rafael, CA), onde um P < 0,05 foi considerado estatisticamente significativo. Resultados: Houve expressão do alfa7nAChR e M3 no homogenato de pulmão de animais controle e sensibilizados. Determinamos por meio da redução de eosinófilos que a dose de 0,5 mg/Kg do tratamento com PNU foi a mais efetiva. Assim, observamos que o tratamento com PNU0,5 nos animais sensibilizados reduziu o número de células totais, eosinófilos, neutrófilos, macrófagos e linfócitos no LBA, assim como número de eosinófilos no sangue periférico e ao redor das vias aéreas. O tratamento com PNU reduziu os níveis de IgE no sangue e as citocinas IL-4, IL-13 e IL-17 no LBA. Todos estes efeitos foram revertido com o pré-tratamento com MLA, exceto para a citocina IL-17. Alem disso, o tratamento com PNU reduziu o remodelamento brônquico (área de edema, de epitélio e de músculo liso e deposição de fibras colágenas) assim como o número de células positivas para MMP-9 e TIMP-1 ao redor das vias aéreas. No pulmão a expressão do p-65-NF-kB, STAT3 fosforilado e o SOCS3 foram inibidas pelo PNU. Conclusão: Estes dados claramente demonstram que o alfa7nAChR está envolvido no controle da resposta inflamatória pulmonar alérgica e de remodelamento brônquico em modelo experimental de asma alérgica e portanto é um novo alvo com potencial terapêutico a ser explorado na fisiopatologia da asma brônquica / Background: Inflammation is one of the most important features in asthma pathophysiology, characterized by eosinophilic response with production of Th2 cytokine profile. The persistence of this inflammation can induce a lung repair process associated with a progressive reduction in lung function, which is not always reversed by available treatments. The anti-inflammatory cholinergic system was described as a neural mechanism that suppresses the immune response and controls inflammation mainly by the activaction of acetylcholine alfa7 nicotinic receptors (alfa7nAChR) found on immune cells. Acetylcholine (ACh) is an important mediator in asthma and we recently demonstrated that partial reduction on ACh release induced lung inflammation per se. Although it is known that muscarinic receptors (mAChRs) has a pro-inflammatory action and causes bronchoconstriction in asthma, the activation of nicotinic receptors (nAChRs) could have a beneficial effect reducing pulmonary inflammation as demonstrated in models of acute and systemic inflammation. The effects of alfa7nAChR activation in the pathophysiology of asthma have not been clearly elucidated. Aim: To investigate the effects of PNU- 282987 (PNU) treatment, a specific alfa7nAChR agonist, in a murine model of chronic allergic airway inflammation. Methods: BALB/c mice were subjected to a protocol of chronic allergic inflammation induced by intraperitoneal ovalbumin (OVA) or saline and subsequent challenges with inhalation. From the 22th day, the animals daily received PNU or vehicle (Ve) until the 28th day. PNU were tested in three differents doses (0.5, 1.0 and 2.0 mg/kg). In order to demonstrate that the effects obtained by PNU treatment was dependent on alfa7nAChR, a group of animals was treated with MLA (antagonist of alfa7nAChR) prior to the PNU treatment. On the 29th day of the protocol, the animals were euthanised and the number of inflammatory cells in the bronchoalveolar lavage fluid(BALF) fluid and blood, cytokine levels in BALF, the expression of alfa7nAChR and mAChRs type 3 (M3), and activation of nuclear transcription factor kB (NF-kB) in the lung were evaluated. Bronchial remodeling was assessed by morfometric methods. Statistical analyses were performed using the SigmaStat (Jandel Scientific, San Rafael, CA) and P < 0.05 is considered statistically significant. Results: ?7nAChR and M3 expression was detected in control and sensitized lung homogenate. The most effective dose of PNU treatment was 0.5 mg/kg evaluated by the effects on eosinophil reduction. Thus, we observed that treatment with PNU0,5 reduced the number of total cells, eosinophils, neutrophils, macrophages and lymphocytes in BALF, as well as number of eosinophils in peripheral blood and around the airways of sensitized animals. The treatment with PNU also reduced IgE levels in the blood, and cytokines IL-4, IL-13 and IL-17 in BALF. All these effects were reversed by pretreatment with MLA, except for IL-17 cytokine. Furthermore, treatment with PNU reduced bronchial remodeling (edema, epithelium and smooth muscle area and airway collagen deposition) as well as the number of positive cells for MMP-9 and TIMP-1 around the airways. The lung p-65-NF-kB, phosphorylated STAT3 and the SOCS3 expression were inhibited by PNU-282987. Conclusion: These data clearly demonstrate that the alfa7nAChR is involved in the control of allergic pulmonary inflammatory response and in bronchial remodeling in an experimental model of allergic asthma and it can be a new target with therapeutic potential to be explored in the pathophysiology of asthma

Les macrophages jouent un rôle dans l'inflammation de certaines pathologies pulmonaires comme l'asthme et la broncho pneumopathie chronique obstructive. Selon la dichotomie Th1 et Th2, les macrophages s'activent en phénotype M1/M2 en fonction du microenvironnement. Sous l'influence du lipopolysaccharide (LPS) les macrophages s'activent en phénotype M1. A l'inverse, l'exposition aux cytokines Th2 (interleukine (IL)-4/IL-13) induit un phénotype M2 des macrophages. Nous avons réalisé une étude transcriptomique des marqueurs de la polarisation M1/M2 des macrophages pulmonaires humains. La polarisation M1 induite par le LPS augmente la production des cytokines (TNF-α, IL-1β, CCL2, 3, 4, 5, CXCL1, 8, 10), de la PGE2 et l'expression du CD38 et CD197. La polarisation M2 induite par l'IL-4/IL-13 augmente l'expression des cytokines (CCL13, 17, 22, 26), de la 15-lipoxygénase (15-LOX) et du CD206. Nous avons évalué l'expression des 15-LOX-1 et 15-LOX-2 et leur rôle dans la régulation de la polarisation des macrophages pulmonaires. Le LPS augmente l'expression de la 15-LOX-2 alors que l'IL-4/IL-13 augmente l'expression de la 15-LOX-1. L'inhibition des 15-lipoxygénases diminue la production des cytokines M1/M2. Enfin, nous avons étudié l'expression et le rôle du récepteur nicotinique α7 dans la polarisation des macrophages pulmonaires humains. Ces derniers expriment les récepteurs nicotiniques α7 dont la stimulation par des agonistes nicotiniques α7 diminue la production des cytokines M1/M2. Ce travail apporte de nouvelles connaissances sur la polarisation des macrophages, dont certaines voies de régulation peuvent être impliquées dans les pathologies inflammatoire pulmonaires / In pulmonary diseases such as asthma and chronic obstructive pulmonary disease, macrophages orchestrate inflammatory reactions. In response to environmental signals, macrophages exhibit a phenotypic polarization that mirrors the Th1/Th2 polarization. Upon exposure to bacterial lipopolysaccharide (LPS), macrophages undergo M1 polarization. In contrast, interleukin (IL)-4/IL-13 induce M2 polarization.In our first study, we characterized the phenotypic differentiation of human lung macrophages (LM) using a whole-transcriptome approach. Cytokines, lipid metabolism and membrane markers were among the most affected genes. LPS-induced M1 polarization was associated with an increase in the production of cytokines (TNF-α, IL-1β, CCL2, 3, 4, 5, CXCL1, 8, 10), in PGE2 signalling and in the expression of CD38 and CD197. IL-4/IL-13-induced M2 macrophages increased expression of cytokines (CCL13, 17, 22, 26), 15-lipoxygenase (15-LOX) and CD206. In the second study, we investigated the expression of 15-LOX-1 and 15-LOX-2 and their roles in regulating the polarization of human LM. LPS increased the expression of 15-LOX-2 whereas IL-4/IL-13 induced the expression of 15-LOX-1. Inhibition of the 15-LOX pathways decreased the production of both M1 and M2 cytokines. The third study investigated the expression of α7 nicotinic receptors (α7nAChR) and their regulating roles in the polarization of LM. Expression of α7nAChR was found in unstimulated LM. Specific α7nAChR agonists decreased the in vitro production of both M1 and M2 cytokines. Our work adds new insights in the macrophage polarization and some of the regulatory pathways that may be involved in pulmonary diseases

Introdução: A inflamação constitui um dos fatores mais importantes da fisiopatologia da asma brônquica, caracterizada por uma resposta eosinofílica com produção de citocinas de perfil Th2. A persistência desta inflamação induz no pulmão um processo de reparo associado à redução progressiva da função pulmonar, que nem sempre é revertida pelos tratamentos disponíveis. O sistema colinérgico anti-inflamatório é descrito como um mecanismo neural que suprime a resposta imune e controla a inflamação principalmente pelo efeito da acetilcolina em receptores nicotínicos do tipo alfa7 (alfa7nAChR) encontrados em células do sistema imune. A acetilcolina (ACh) exerce um importante efeito na asma e recentemente demonstramos que a redução parcial da liberação da acetilcolina induz per se a inflamação pulmonar. Embora se saiba que os receptores muscarínicos (mAChRs) exercem um efeito pró-inflamatório e broncoconstritor na asma, a ativação de receptores nicotínicos (nAChRs) poderia ter um efeito benéfico reduzindo a inflamação pulmonar, fato demonstrado em modelos de inflamação sistêmica e aguda. O efeito da ativação do alfa7nAChR na fisiopatologia da asma ainda não está claramente elucidado. Objetivo: Investigar o efeito do tratamento com PNU-282987 (PNU), um agonista específico do alfa7nAChR, em um modelo murino de inflamação alérgica crônica das vias aéreas. Metodologia: Camundongos BALB/c foram submetidos ao protocolo de indução alérgica crônica das vias aéreas com ovoalbumina (OVA) ou salina intraperitoneal (i.p.) e posterior desafios inalatórios. A partir do 22° dia, os animais receberam diariamente tratamento com PNU ou veículo (Ve) até o 28° dia. Foram testadas três doses de PNU (0,5, 1,0 e 2,0 mg/Kg). A fim de evidenciar se o efeito obtido no tratamento com PNU era dependente do receptor alfa7nAChR, um grupo de animais foi tratado com MLA (antagonista do alfa7nAChR), previamente ao tratamento com PNU. No 29° dia do protocolo, os animais foram eutanasiados e foram avaliados o número de células inflamatórias no lavado broncoalveolar (LBA) e no sangue, os níveis de citocinas no LBA, a expressão do alfa7nAChR e mAchRs do tipo 3 (M3) e a ativação do fator de transcrição nuclear kB (NF-kB) no pulmão. O remodelamento brônquico foi avaliado por morfometria. As análises estatísticas foram realizadas por meio do programa SigmaStat (Jandel Scientific, San Rafael, CA), onde um P < 0,05 foi considerado estatisticamente significativo. Resultados: Houve expressão do alfa7nAChR e M3 no homogenato de pulmão de animais controle e sensibilizados. Determinamos por meio da redução de eosinófilos que a dose de 0,5 mg/Kg do tratamento com PNU foi a mais efetiva. Assim, observamos que o tratamento com PNU0,5 nos animais sensibilizados reduziu o número de células totais, eosinófilos, neutrófilos, macrófagos e linfócitos no LBA, assim como número de eosinófilos no sangue periférico e ao redor das vias aéreas. O tratamento com PNU reduziu os níveis de IgE no sangue e as citocinas IL-4, IL-13 e IL-17 no LBA. Todos estes efeitos foram revertido com o pré-tratamento com MLA, exceto para a citocina IL-17. Alem disso, o tratamento com PNU reduziu o remodelamento brônquico (área de edema, de epitélio e de músculo liso e deposição de fibras colágenas) assim como o número de células positivas para MMP-9 e TIMP-1 ao redor das vias aéreas. No pulmão a expressão do p-65-NF-kB, STAT3 fosforilado e o SOCS3 foram inibidas pelo PNU. Conclusão: Estes dados claramente demonstram que o alfa7nAChR está envolvido no controle da resposta inflamatória pulmonar alérgica e de remodelamento brônquico em modelo experimental de asma alérgica e portanto é um novo alvo com potencial terapêutico a ser explorado na fisiopatologia da asma brônquica / Background: Inflammation is one of the most important features in asthma pathophysiology, characterized by eosinophilic response with production of Th2 cytokine profile. The persistence of this inflammation can induce a lung repair process associated with a progressive reduction in lung function, which is not always reversed by available treatments. The anti-inflammatory cholinergic system was described as a neural mechanism that suppresses the immune response and controls inflammation mainly by the activaction of acetylcholine alfa7 nicotinic receptors (alfa7nAChR) found on immune cells. Acetylcholine (ACh) is an important mediator in asthma and we recently demonstrated that partial reduction on ACh release induced lung inflammation per se. Although it is known that muscarinic receptors (mAChRs) has a pro-inflammatory action and causes bronchoconstriction in asthma, the activation of nicotinic receptors (nAChRs) could have a beneficial effect reducing pulmonary inflammation as demonstrated in models of acute and systemic inflammation. The effects of alfa7nAChR activation in the pathophysiology of asthma have not been clearly elucidated. Aim: To investigate the effects of PNU- 282987 (PNU) treatment, a specific alfa7nAChR agonist, in a murine model of chronic allergic airway inflammation. Methods: BALB/c mice were subjected to a protocol of chronic allergic inflammation induced by intraperitoneal ovalbumin (OVA) or saline and subsequent challenges with inhalation. From the 22th day, the animals daily received PNU or vehicle (Ve) until the 28th day. PNU were tested in three differents doses (0.5, 1.0 and 2.0 mg/kg). In order to demonstrate that the effects obtained by PNU treatment was dependent on alfa7nAChR, a group of animals was treated with MLA (antagonist of alfa7nAChR) prior to the PNU treatment. On the 29th day of the protocol, the animals were euthanised and the number of inflammatory cells in the bronchoalveolar lavage fluid(BALF) fluid and blood, cytokine levels in BALF, the expression of alfa7nAChR and mAChRs type 3 (M3), and activation of nuclear transcription factor kB (NF-kB) in the lung were evaluated. Bronchial remodeling was assessed by morfometric methods. Statistical analyses were performed using the SigmaStat (Jandel Scientific, San Rafael, CA) and P < 0.05 is considered statistically significant. Results: ?7nAChR and M3 expression was detected in control and sensitized lung homogenate. The most effective dose of PNU treatment was 0.5 mg/kg evaluated by the effects on eosinophil reduction. Thus, we observed that treatment with PNU0,5 reduced the number of total cells, eosinophils, neutrophils, macrophages and lymphocytes in BALF, as well as number of eosinophils in peripheral blood and around the airways of sensitized animals. The treatment with PNU also reduced IgE levels in the blood, and cytokines IL-4, IL-13 and IL-17 in BALF. All these effects were reversed by pretreatment with MLA, except for IL-17 cytokine. Furthermore, treatment with PNU reduced bronchial remodeling (edema, epithelium and smooth muscle area and airway collagen deposition) as well as the number of positive cells for MMP-9 and TIMP-1 around the airways. The lung p-65-NF-kB, phosphorylated STAT3 and the SOCS3 expression were inhibited by PNU-282987. Conclusion: These data clearly demonstrate that the alfa7nAChR is involved in the control of allergic pulmonary inflammatory response and in bronchial remodeling in an experimental model of allergic asthma and it can be a new target with therapeutic potential to be explored in the pathophysiology of asthma

A nicotina é um alcalóide extraído da planta do tabaco e age como agonista em neurônios e outras células que expressam receptores nicotínicos. Essa substância é capaz de atravessar a barreira hematoencefálica e mimetizar os efeitos da acetilcolina endógena ao interagir com os diferentes subtipos de receptores. O hipocampo e o cerebelo são regiões encefálicas ricas em receptores nicotínicos como o alfa7 e o alfa42 que estão também envolvidos na progressão de doenças neurodegenerativas, como a doença de Alzheimer, e neuropsiquiátricas, como a esquizofrenia, ambas apresentando um contexto inflamatório. O lipopolissacarídeo (LPS) é um indutor inflamatório que age via receptor toll-4 (TLR-4) ativando, dentre outros, o fator de transcrição NF-capaB. Vários estudos têm demonstrado tanto in vitro quanto in vivo que a exposição crônica à nicotina inibe a resposta imune. Uma vez que o fator de transcrição NF-capaB regula a expressão gênica de diversos fatores que medeiam a plasticidade e o desenvolvimento neuronal e inflamação, nosso objetivo foi avaliar os efeitos da nicotina crônica sobre a via de sinalização induzida pelo LPS. Sendo assim, avaliou-se a translocação nuclear do NF-capaB, a expressão de indutores inflamatórios (TNF, IL-1 e iNOS) e também do receptor TLR-4 tanto no hipocampo quanto no cerebelo. Além disso, foram determinados os níveis plasmáticos das citocinas TNF, IL-1, MIP-1alfa, CINC2alfa/. Nossos resultados mostram que, perifericamente, o LPS induziu a produção das citocinas plasmáticas, mas a nicotina não foi capaz de reverter seus efeitos. Já no cerebelo e no hipocampo, a nicotina crônica (0,1 e 1,0 mg/kg) interferiu significativamente na translocação do NF-capaB induzida pelo LPS, levando inclusive a um bloqueio importante da expressão do mRNA de TNF, IL-1, iNOS no hipocampo, e de IL-1 no cerebelo. Ademais, no hipocampo, a nicotina também interferiu na expressão do receptor TLR-4. A mecamilamina, antagonista heteromérico não-seletivo não foi capaz de reverter os efeitos da nicotina sobre o NF-capaB no hipocampo, mas o fez no cerebelo. Já o antagonista seletivo de receptor alfa 7, metililaconitina, bloqueou a ação da nicotina no hipocampo. / Nicotine is an alkaloid extracted from Tobacco plants, and it acts as an agonist in neurons and other cells that express nicotinic receptors. This substance crosses the blood brain barrier and mimics endogenous acetylcholine in interacting with various receptor subtypes. The hippocampus and cerebellum are brain regions rich in nicotinic receptors including 7, 42 and other subtypes, involved in neurodegenerative and neuropsychiatric disorders, including Alzheimer disease and schizophrenia, respectively, in a neuroinflammatory context. The lipopolysaccharide (LPS) is a known inflammatory inducer that exerts its effects through the Toll-like receptor 4 (TLR-4) and activates the transcription factor NF-capaB. In the present work, we evaluate the effects of chronic nicotine on the nuclear translocation of NF-capaB, on the expression of NF-capaB regulated inflammatory inducers (TNF, IL-1 and iNOS) and on the LPS-induced TLR4 mRNA expression in response to chronic nicotine treatment in hippocampus and cerebellum. In addition, we accessed the production of peripheral cytokines (TNF, IL-1, MIP-1, CINC 2/). Our results demonstrate that LPS induced the production of peripheral cytokines but nicotine treatment did not interfere in their plasma levels. However, in cerebellum and hippocampus, chronic nicotine 1.0 and 0.1 mg/kg interfered in the LPS-induced NF-capaB leading to a deficient mRNA expression of its related genes TNF, IL-1 and iNOS in hippocampus and of IL-1, in cerebellum. Chronic nicotine (0.1 mg/kg, s.c.) significantly interfered in the TLR4 mRNA expression in LPS-challenged animal group in hippocampus. The non-selective heteromeric antagonist mecamylamine (1.0 mg/kg s.c.) reverted the nicotine effects over NF-capaB nuclear translocation in cerebellum, but not in hippocampus. On the other hand, the 7 antagonist methyllycaconitine (MLA) (5.0 mg/kg i.p.) reverted the nicotine effects over NF-capaB in hippocampus but not in cerebellum. Nicotine prevents LPS effects, both in cerebellum and hippocampus, once it reduced significantly the nuclear translocation of NF-capaB and the expression of related pro-inflammatory cytokines. Besides, the nicotinic effects seem to be mainly mediated by heteromeric receptors in cerebellum and through 7 receptors in hippocampus.

Palmoplantar pustulosis (PPP) is a chronic disease of unknown pathogenesis. Most of the patients were smokers. High prevalence of a number of autoimmune diseases was observed among the patients (thyroid disease 14%, gluten intolerance 8%, diabetes type 1 3%). Eosinophils and neutrophils were found in large numbers in the pustules. Massive infiltrates of lymphocytes and mast cells in the dermis below the pustule and an abnormal acrosyringial pattern indicate that the acrosyringium is the target for the inflammation. Immunofluorescence (IF) revealed decreased innervation of the sweat gland, outward migration of substance P-positive granulocytes in the acrosyringium and an increased number of contacts between mast cells and nerve fibres in the dermis. Distributions of choline acetyltransferase (ChAT) and acetylcholinesterase (AChE) were studied, since they regulate the level of acetylcholine, the main inducer of sweating. The most intense AChE-like immunoreactivity (LI) was observed in the acrosyringium in the lowest part of the stratum corneum, corresponding to the site of the pustule in PPP. ChAT-LI in granulocytes and AChE-LI in mast cells were demonstrated, which may have implications for inflammatory processes in general. Nicotinic acetylcholine receptors (nAChR) are activated by acetylcholine but also by nicotine. Immunohistochemstry of α-3 and α-7 subtypes of the nAChRs showed that the nAChR expression in healthy skin was influenced by smoking. A highly abnormal α-7 nAChR distribution in PPP skin was observed. The levels of nAChR antibodies were elevated in 42% of the PPP sera, and 68% of these sera gave specific endothelial IF in the papillary dermis in skin from non-smokers. Positive IF in the acrosyringium was also noted in skin from smokers. Conclusions: Smoking seems to induce up-regulation of an antigen in palmar skin. The results indicate that PPP is an autoimmune disease and that nicotine might have a role in the onset of the inflammation.