Les peptides antimicrobiens (PAMs) cationiques sont des molécules amphipatiques conservées chez une grande diversité d'espèces vivantes. Ils participent ainsi à la défense immunitaire de nombreux organismes incluant les bactéries, les insectes, les plantes et les vertébrés. En plus de leur activité microbicide directe dirigée contre un large spectre de pathogènes, la plupart des PAMs cationiques sont désormais connus pour exercer des fonctions immunomodulatrices sur les réponses innée et adaptative. Notre équipe a récemment découvert et isolé un nouveau PAM à partir du venin de la fourmi Tetramorium bicarinatum, nommé P17. Dans ce travail, nous avons étudié les propriétés immunomodulatrices du P17 sur la réponse immunitaire innée médiée par les macrophages. Nous nous sommes plus particulièrement intéressés à sa capacité à moduler la différenciation de macrophages dérivés de monocytes humains (h-MDM) ainsi que leurs fonctions fongicides associées vis-à-vis d'une levure opportuniste majeure Candida albicans (C. albicans). Nous avons ainsi pu mettre en évidence que le P17 oriente la différenciation des h-MDM vers un phénotype alternatif caractérisé par la surexpression des récepteurs lectine de type C (CLRs) tels que Dectine-1 et le récepteur mannose (MR). De manière intéressante, nous avons mis en évidence que la surexpression de ces deux récepteurs à la surface des h-MDM activés par le P17 nécessite la mobilisation de l'acide arachidonique et la production de leucotriène B4 (LTB4). Nous avons également démontré que ce métabolite de l'AA conduit à l'activation du récepteur nucléaire PPARƴ, facteur clé de l'activation alternative des macrophages et de l'expression des CLRs associée à ce phénotype. Au cours de ce travail, nous avons démontré que les h-MDM polarisés par le P17 présentent une meilleure capacité à éliminer C. albicans. En effet, ces h-MDM activés par le P17 ont une capacité de reconnaissance, par les CLRs Dectine-1 et MR, et de phagocytose de C. albicans augmentée. De plus, l'étude des mécanismes microbicides conduisant à l'élimination de C. albicans révèle que les h-MDM activés par le P17 produisent de fortes quantités d'espèces réactives de l'oxygène (ROS) et d'IL-1ß via l'inflammasome. Ainsi, ce travail met en évidence que l'induction de l'activité fongicide des h-MDM par le P17 est dépendante de l'axe LTB4/ PPARƴ/Dectine-1-MR. Nous avons finalement confirmé ces données in vivo sur un modèle de candidose gastro-intestinale induite chez des souris traitées par voie intra-péritonéale par P17 ou non. Les résultats obtenus ont révélé que les souris traitées par P17 étaient plus résistantes à l'infection gastro-intestinale à C. albicans. La diminution de la charge fongique au niveau du cæcum des souris traitées par le P17 est associée à une meilleure efficacité de leurs macrophages à phagocyter C. albicans, à produire des ROS et à tuer C. albicans. Ainsi, ces résultats identifient le P17 comme un activateur original des propriétés antifongiques des macrophages agissant en aval de la voie permettant l'induction de l'expression des CLRs via PPARƴ. Ces données révèlent pour la première fois l'implication d'un PAM dans le contrôle de la différenciation des macrophages et leurs fonctions microbicides. / Cationic antimicrobial peptides (AMPs) are evolutionary small and amphipatic conserved molecules which are involved in the immune defense of a wide range of organisms, including bacteria, insects, plants and vertebrates. Beside their direct microbicidal activity against pathogens, most of them are known to exert immunomodulatory functions on innate and adaptive immune cells. Here we evaluated the immunomodulatory properties of an original cationic AMP, named P17, discovered and isolated by our team from the ant Tetramorium bicarinatum venom. We have focused on its efficiency to modulate human monocyte-derived macrophages (h-MDM) differentiation and its capacity to provide them an antifungal activity against the main opportunistic yeast Candida albicans (C. albicans). We showed that P17 directed h-MDM polarization toward an alternative phenotype characterized by mannose (MR) and dectin-1 C-type lectin receptors (CLRs) upregulation. Interestingly, we demonstrated that this upregulation of MR and Dectin-1 in P17-treated h-MDM requires AA mobilization and leukotriene B4 (LTB4) synthesis, essential for PPAR activation. We also demonstrated that this AA metabolite led to the PPARƴ nuclear receptor activation which is a key factor of macrophages alternative activation and the associated CLRs expression. In this study, we observed that P17-activated h-MDM exhibited an improved capacity to eliminate C. albicans. Indeed, these P17-polarized macrophages displayed an increased ability to recognize and phacocyte yeasts. Furthermore, the study of microbicidal mechanisms leading to C. albicans clearance revealed that P17-activated h-MDM produced reactive oxygen species (ROS) and inflammasome-dependant IL-1ß in high amounts. These mechanisms induction in P17-polarized h-MDM was dependent on the LTB4/ PPARƴ/Dectin-1-MR axis. Finally, these data were supported by in vivo experiments demonstrating that P17-treated mice infected with C. albicans developed less severe gastrointestinal infection related to a higher efficiency of their macrophages to engulf C. albicans, to produce ROS and to kill yeasts. Altogether, these results identify P17 as an original activator of the fungicidal response of macrophages that acts downstream the pathway leading to CLRs expression through PPARƴ activation.

Francisella tularensis, l'agent de la tularémie, est une bactérie intracellulaire capable d'infecter un grand nombre de cellules dont les macrophages. Le système immunitaire inné cytosolique est capable de détecter la bactérie à différents stades de son cycle d'infection. Dans un premier temps, le macrophage détecte la bactérie cytosolique et produit de l'interféron de type I. Cet interféron induit l'expression de milliers de gènes. Le macrophage est ensuite capable de détecter l'ADN cytosolique de la bactérie via un récepteur spécifique AIM2. La liaison AIM2-ADN entraine la formation d'un complexe multi-protéique appelé inflammasome et se composant de AIM2-ASC-caspase-1. L'activation de ce complexe conduit à la maturation de la caspase-1. Caspase-1 permet la sécrétion de deux cytokines majeures antimicrobiennes : l'IL-1beta et l'IL-18. De plus, caspase-1 induit une mort programmée des cellules infectées appelée pyroptose. La sécrétion de cytokines et la pyroptose sont deux évènements majeurs pour lutter contre les pathogènes. Ma thèse a consisté à identifier le lien entre l'interféron et l'activation de l inflammasome AIM2 dans des macrophages infectés par la bactérie Francisella. En réalisant un crible a l'aide d'ARNs interférents, j'ai découvert que 2 protéines sont impliquées dans l'activation de cet inflammasome, les guanylate binding proteins 2 et 5 (GBP2 et GBP5). En collaboration avec l'équipe du Dr. Broz en Suisse, nous avons démontré que les GBPs étaient impliquées dans le contrôle de la réplication intracellulaire de Francisella et également dans la lyse de la bactérie permettant le relargage d'ADN et l'activation de l'inflammasome AIM2. Les GBPs sont induites par l'interféron de type I mais très majoritairement par l'interféron de type II (IFN- gamma). Nous avons mis en évidence que le contrôle de la réplication bactérienne est GB dépendant et inflammasome-dépendant en absence d'IFN- gamma mais qu'il devient totalement GB dépendant et inflammasome-indépendant dans des macrophages pré-stimulés avec de l'IFN- gamma. De plus, la mort des macrophages pré-stimulés avec de l'IFN- gamma et infectés par Francisella est également GBP-dépendante et inflammasome-indépendante. En prenant en compte tous ces résultats, nous concluons que les GBPs sont des protéines impliquées dans l'immunité des macrophages infectés par Francisella mais qu'elles ont un double rôle : d'une part celui d'induire l'activation de l'inflammasome (la pyroptose) sous le contrôle de l'interféron de type I et d'autre part, d'induire une mort cellulaire et la lyse des bactéries cytosoliques de manière indépendante de l'inflammasome sous le contrôle d'IFN- gamma. Nos résultats placent donc les GBPs comme les effecteurs majeurs de l'immunité cytosolique antibactérienne suite au traitement par l'IFN-gamma / Francisella tularensis is an intracellular bacterium, and the causative agent of tularemia, capable of infecting a large number of cells including macrophages. The innate cytosolic immune system is capable of detecting the bacterium at different stages of its infection cycle. Macrophages first detect the DNA of the cytosolic bacterium and produce type I interferon. Type I interferon subsequently induces the expression of thousands of genes. The macrophages then detect the cytosolic DNA of the bacterium via a cytosolic DNA sensor called AIM2. The AIM2-DNA binding results in the formation of a multi-protein complex called the AIM2 inflammasome composed of AIM2-ASC-caspase-1. Activation of this complex leads to the maturation of caspase-1. Caspase-1 activation leads to the secretion of two major antimicrobial cytokines, IL-1ß and IL-18. In addition, caspase-1 induces a programmed cell death termed pyroptosis. Cytokine secretion and pyroptosis are two major events in the control of pathogens. My PhD focused in identifying the link between interferon and activation of the AIM2 inflammasome in macrophages infected with the pathogenic bacterium Francisella. I performed a RNA interference screening and identified two proteins involved in the activation of the AIM2 inflammasome: guanylate binding proteins 2 and 5 (GBP2 and GBP5). In collaboration with Dr. Broz’s team in Switzerland, we demonstrated that GBPs are involved in the control of intracellular replication of Francisella and also in the lysis of the bacterium allowing the release of bacterial DNA and the activation of inflammasome AIM2. GBPs are induced by type I interferon but to a much greater extent by type II interferon (IFN-gamma). In the second part of my work, we demonstrate that the control of bacterial replication is GBP-dependent and inflammasome-dependent in the absence of IFN-gamma but that it becomes fully GBP-dependent and inflammasome-independent in macrophages primed with IFN-gamma. Cell-death of macrophages primed with IFN-? and infected with Francisella is also GBP-dependent and inflammasome-independent. Taken together, these results demonstrate that GBPs are innate immunity proteins involved in the death of macrophages and the bacterial growth restriction through two differents pathways : one induces the activation of inflammasome (induction of Pyroptosis) controlled with type I interferon signaling and, another induces cell-death and bacterial killing in an inflammasome-independent manner under the control of IFN-gamma. Our results thus discriminates the antimicrobial action of the inflammasome and of GBPs and position GBPs as the master antibacterial effectors of IFN-gamma, a key cytokine to fight cytosolic bacteria

Les inflammasomes sont des complexes protéiques intracellulaires qui ont un rôle majeur dans l'immunité innée. Leur activation conduit à la mort de la cellule dans un contexte hyperinflammatoire. Compte-tenu des effets potentiellement délétères, tissulaires et systémiques, les inflammasomes sont strictement régulés. A l'heure actuelle, la compréhension des mécanismes conduisant à leur activation et leur régulation reste partielle. Dans une première partie de cette thèse, nous avons utilisé une technique de biotinylation proximale (BioID) pour identifier les protéines interagissant avec l'inflammasome. Nous avons identifié 111 protéines dont la relation étroite avec l'inflammasome était vraisemblable. Parmi ces 111 protéines, 25% avaient d'ailleurs déjà été décrites comme des protéines interagissant avec le complexe. L'identification d'un adaptateur majeur de l'autophagie, p62/sequestosome-1 (p62), nous a conduit à focaliser notre attention sur son rôle dans la régulation de l'inflammasome. Nous avons d'abord démontré que l'interaction entre p62 et l'inflammasome existait, sur le plan biochimique. Par la suite, nous avons prouvé que p62 était un substrat du complexe et que l'activation de ce dernier entrainait le clivage de p62 au niveau d'un résidu aspartique en position 329. Enfin, nous avons caractérisé les conséquences fonctionnelles de ce clivage, en montrant que les fragments protéiques générés entrainaient une régulation positive ou négative du complexe. Nous avons alors émis l'hypothèse que p62 pourrait réguler l'inflammasome de manière différente selon le signal activateur. Dans une seconde partie, translationnelle, nous nous sommes intéressés aux conséquences des mutations dans la séquence de gènes codant les constituants de l'inflammasome ou des protéines régulatrices du complexe. Celles-ci sont à l'origine des maladies auto-inflammatoires monogéniques. Ces maladies sont caractérisées par des épisodes récurrents de fièvre associés variablement à d'autres symptômes systémiques. La plus fréquente est la fièvre méditerranéenne familiale (FMF), avec une prévalence estimée entre 1 et 5 pour 10 000 habitants en France. Les mutations du gène MEFV, codant la pyrine, sont à l'origine de la FMF. La pyrine peut induire la formation d'un inflammasome spécifique. Récemment, le mécanisme d'activation de l'inflammasome pyrine a été mieux caractérisé : certaines toxines (comme la toxine B du Clostridium difficile, TcdB) induisent l'activation de l'inflammasome pyrine. Nous avons utilisé ces nouvelles connaissances afin d'explorer les conséquences de l'activation de l'inflammasome pyrine par la TcdB dans les monocytes des patients atteints de FMF, comparés aux monocytes de donneurs sains. Nos résultats indiquent que ces mutations induisent un abaissement du seuil d'activation de l'inflammasome pyrine. Par ailleurs, les corrélations génotype/phénotype indiquent qu'il existe un effet de dosage génétique, en lien avec le nombre d'allèles mutés. Ces résultats ouvrent de nouvelles perspectives pour les patients atteints de FMF, tant dans la compréhension de la physiopathologie que dans la possibilité de mise au point de tests fonctionnels pour le diagnostic de la maladie / Inflammasomes are intracellular multiprotein complexes that have a major role in innate immunity. Their activation leads to hyperinflammatory cell death. In view of potentially deleterious effects, the inflammasomes are strictly regulated. At present, the understanding of the mechanisms leading to their activation and regulation remains partial. In a first part of this thesis, we used a technique of proximity-dependent biotinylation (BioID) to identify the proteins interacting with the inflammasome. We identified 111 proteins with a close relationship to the inflammasome. Among these 111 proteins, 25% had already been described as proteins interacting with the complex. The identification of a major adaptor of autophagy, p62/sequestosome-1 (p62), led us to focus our attention on its role in the regulation of inflammasome. We first demonstrated that the interaction between p62 and inflammasome was real, at the biochemical level. Subsequently, we proved that p62 was a substrate of the complex and that the activation of the latter led to the cleavage of p62 at the aspartate 329. Finally, we characterized the functional consequences of this cleavage and showed that the protein fragments generated led to a positive or negative regulation of the complex. We thus hypothesized that p62 could regulate the inflammasome differently according to the activator signal. In a second translational part, we looked at the consequences of mutations in the sequence of genes coding components of the inflammasome or proteins regulating it. These are the cause of monogenic auto-inflammatory diseases. These diseases are characterized by recurrent episodes of fever associated with other systemic symptoms. The most frequent is Familial Mediterranean Fever (FMF), with prevalence estimated at between 1 and 5 per 10 000 inhabitants, in France. Mutations of the MEFV gene, encoding pyrin, cause FMF. Pyrine may trigger the formation of a specific inflammasome. Recently, the mechanism of activation of the pyrin inflammasome has been better characterized: toxins (such as toxin B of Clostridium difficile, TcdB) induce the activation of the pyrin inflammasome. We used this new knowledge to investigate the consequences of TcdB on pyrin inflammasome activation in monocytes from FMF patients compared to monocytes from healthy donors. Our results indicate that these mutations induce a decreased threshold of activation of the pyrin inflammasome. In addition, genotype / phenotype correlations indicate a gene-dosage effect, related to the number of mutated alleles. These results open new perspectives for patients with FMF, in understanding the pathophysiology of the diseass and in developing functional diagnostic tests

La pandémie de l'obésité entraine une augmentation de la prévalence des maladies chroniques du foie ou stéatopathies métaboliques (NAFLD). Le spectre des NAFLD va de la stéatose caractérisée par une accumulation de lipides dans le foie à la stéatohépatite (NASH) associant une inflammation, de la mort hépatocytaire et de la fibrose. Lors de l'obésité, l'élévation de signaux de dangers métaboliques perturbe les fonctions du réticulum endoplasmique (RE) essentielles pour l’homéostasie cellulaire. Les perturbations sont transmises par 3 senseurs : IRE1α, ATF6 et PERK pour activer une réponse adaptative. Si ce stress est sévère ou devient chronique, la cellule enclenchera une réponse terminale apoptotique. La protéine Bax Inhibitor-1 (BI-1) pourrait jouer un rôle hépatoprotecteur en inhibant l’hyperactivation de la voie de signalisation IRE1α.En combinant des études chez l’homme et dans des modèles animaux, l’objectif de cette étude était de mieux caractériser l'activation chronique du stress du RE dans les NAFLD. Ce travail a émis l’hypothèse qu’une déficience en BI-1 entrainerait l’activation soutenue de la voie IRE1α qui serait responsable de la transition de la stéatose à la NASH. Cette étude s'intéresse au dialogue potentiel entre le stress du RE et l’activation de l'inflammasome NLRP3, qui induit la sécrétion des cytokines pro-inflammatoires (IL-1β, IL-18) grâce aux caspases pro-inflammatoires (caspase-1, caspase-4/11). L’utilisation d’un inhibiteur global du stress du RE ou des inhibiteurs pharmacologiques spécifiques à la voie IRE1α améliorerait les caractéristiques pathophysiologiques de la NASH et pourrait ouvrir de nouvelles perspectives thérapeutiques. / Due to the obesity pandemic, the last decades have been marked by a constantly increasing prevalence of Non-Alcoholic Fatty Liver Disease (NAFLD). NAFLD covers a spectrum of hepatic disorders ranging from steatosis, characterized by the ectopic accumulation of lipids in the liver, to steatohepatitis (NASH), featuring inflammation, hepatocellular death and fibrosis. During obesity, an increase in metabolic danger signals leads to disrupted endoplasmic reticulum (ER) function, essential for cellular homeostasis. The resulting ER stress activates a signaling network involving three sensors: IRE1α, ATF6 and PERK to enforce adaptive programs. If this stress is severe or becomes chronic, the cell will trigger a terminal apoptotic response. The protein Bax Inhibitor-1 (BI-1), as a negative endogenous regulator of the IRE1α signaling pathway in the liver, may play a hepatoprotective role.By combining data from obese patients with liver complications and experimental approaches in mice, this thesis aimed to better characterize the chronic activation of ER stress in NAFLD pathogenesis. This work also emitted the hypothesis that a deficiency in BI-1 leads to unrestrained IRE1α signaling that may be responsible for the steatosis to NASH transition. This study further investigated the potential dialogue between ER stress and the activation the NLRP3 inflammasome, which induces the secretion of pro-inflammatory cytokines (IL-1β, IL-18) by activating pro-inflammatory caspases (caspase-1, caspase-4/11). The administration of a broad spectrum ER stress inhibitor or specific inhibitors of IRE1α improved the pathophysiological features of NASH and may open novel therapeutic perspectives.

Atherosclerosis is complex inflammatory disease of the arterial wall with progressive accumulation of lipids and narrowing of the vessel. Increasing evidence suggest that inflammation plays an important role in plaque stability and often accelerate cardiovascular events such as myocardial infarction (MI). Among the vast number of inflammatory cytokines, IL-1β is known to be a key modulator in vessel wall inflammation and acceleration of the atherosclerotic process. The biologically active IL-1β is regulated by a multiprotein complex known as the NLRP3 inflammasome complex. In this thesis, we have focused on polymorphisms in the NLRP3 and CARD8 genes and their possible association to atherosclerosis and/or MI. We have also investigated the expression of inflammasome components NLRP3 and CARD8 in atherosclerosis and the role of genetic variants for the expression of these genes. The expression of NLRP3, CARD8, ASC, caspase-1, IL-1β, and IL-18 were found significantly upregulated in atherosclerotic lesions compared to normal arteries. Human carotid plaques not only express the NLRP3 inflammasome, but also release IL-1β upon exposure to lipopolysaccharide (LPS), adenosine triphosphate (ATP) and cholesterol crystals, which suggest NLRP3 inflammasome activation in human atherosclerotic lesions. Also, CARD8 was found to be important in the regulation of several inflammatory markers in endothelial cells, like RANTES, IP10 and ICAM-1. We further assessed the potential association of a CARD8 polymorphism and polymorphisms located downstream of the NLRP3 gene to the risk of MI in two independent Swedish cohorts. The CARD8 variant exhibited no association to risk of MI in either of the two cohorts. Some of the minor alleles of NLRP3 variants were associated with increased IL-1β levels and to NLRP3 mRNA levels in peripheral blood monocytic cells (PBMC). Taken together, the present thesis shows that NLRP3 inflammasome activation and increased expression of CARD8 in the atherosclerotic plaque might be possible contributors to the enhanced inflammatory response and leukocyte infiltration in the pathophysiology of atherosclerosis.

Alzheimer’s disease is a devastating neurodegenerative disorder and the leading cause of dementia. The disease manifests via several pathologies including neuroinflammation, oxidative stress, metal ion dyshomeostasis, and cell death. To address the multifaceted nature of this disorder, the design of several diverse compounds, targeting many pathological effects, was generated. First, a series of compounds based on curcumin and diosgenin were synthesized following the bivalent design strategy. Two compounds were discovered to have neuroprotective ability, anti-oxidative function, and anti-Aß oligomerization (AßO) properties. A second set of molecules was also designed, wherein a hybrid compound strategy was utilized. Three hybrids were to shown to protect MC65 cells from Aß-induced toxicity and to have significant anti-oxidative activity. Mechanistic studies propose that protection is through disruption of interactions between AßOs and partner proteins. Furthermore, one hybrid was also shown to be able to pass the BBB. Lastly, studies of glyburide, an anti-diabetic medication, have shown an off-target anti-inflammatory effect specific for the NLRP3 inflammasome, which has been implicated in AD development. Therefore, a series of glyburide analogs were synthesized and characterized. One analog was able to successfully inhibit the NLRP3 inflammasome and reduce IL-1ß expression without affecting blood glucose. In vivo studies demonstrated an ability to prevent or ameliorate adverse inflammation-related outcomes in murine inflammatory models. Altogether, these investigations have yielded three novel series of compounds, all capable of modifying Alzheimer’s disease pathology. These results warrant future investigations into the development, optimization, and characterization of these analogs as potential treatments for Alzheimer’s disease.

O aumento da concentração sérica de testosterona está associado tanto a fatores de risco cardiovascular, incluindo obesidade abdominal e hipertensão arterial, como diretamente a doenças cardiovasculares (DCVs). Há evidências que a testosterona pode modular, positivamente, componentes envolvidos em processos de oxirredução (redox) e inflamatório, incluindo a geração de espécies reativas de oxigênio (EROs) e produção de citocinas próinflamatórias e anti-inflamatórias. O inflamassoma NLRP3 é um componente do sistema imunológico inato e regulador importante da inflamação crônica. Sua ativação pode ser mediada pelo aumento de EROs, contribuindo para o processo inflamatório presente em diversas DCVs. Considerando que a testosterona representa uma fonte importante na produção de EROs, foi testada a hipótese que níveis suprafisiológicos de testosterona induzem ativação do complexo NLRP3 inflamassoma, com consequente prejuízo da função vascular. Esse estudo avaliou se níveis suprafisiológicos de testosterona são capazes de ativar o inflamassoma NLRP3 e se esta ativação contribui para alterações na reatividade vascular. Nosso estudo demonstrou que níveis supra fisiológicos de testosterona alteraram a função vascular, com participação dos receptores para andrógenos em camundongos C57BL/6J wild type (WT). Estes efeitos da testosterona não foram observados em camundongos WT incubados com MCC950 (inibidor do receptor NLRP3) e knockout NLRP3 (NLRP3- / - ). Além disso, a testosterona aumentou a geração vascular de EROs, determinada pela fluorescência de lucigenina e dihidroetidina. A geração de EROs foi prevenida por cianeto de carbonil mclorofenil hidrazona (CCCP), um desacoplador mitocondrial. A testosterona em níveis suprafisiológicos aumentou a expressão vascular de caspase-1 e interleucina-1? (IL-1?), como determinado por Western Blotting e Elisa, respectivamente. Esses dados sugerem que níveis suprafisiológicos de testosterona induzem disfunção vascular via geração de EROs e ativação do inflamassoma NLRP3 / Increased serum testosterone concentration is associated with both cardiovascular risk factors, including abdominal obesity and hypertension, and cardiovascular disease (CVD). There is evidence that testosterone positively modulates components involved in oxidative and inflammatory processes, including the generation of reactive oxygen species (ROS) and production of pro-inflammatory and anti-inflammatory cytokines. NLRP3 inflammasome is a component of the innate immune system and an important modulator of chronic inflammation. NLRP3 activation can be mediated by increased levels of ROS, contributing to chronic inflammation in several CVDs. Considering that testosterone induces ROS production, we tested tested the hypothesis that supraphysiological levels of testosterone activates the NLRP3 inflammasome, with consequent impairment of vascular function. This study evaluated whether supraphysiological levels of testosterone activate NLRP3 inflammasome and whether NLRP3 activation contributes to testosterone-induced vascular dysfunction. Our study demonstrated that supraphysiological levels of testosterone, via activation of androgen receptors, altered vascular function in C57BL/6J wild type (WT) mice. The vascular effects of testosterone were not observed in WT mice incubation with MCC950 (NLRP3 receptor inhibitor) and NLRP3 (NLRP3 - / - ) knockout mice. In addition, testosterone increased vascular generation of ROS, as determined by lucigenin and dihydroetidine the fluorescence. ROS generation was prevented by carbonyl m-chlorophenyl hydrazone cyanide (CCCP), a mitochondrial uncoupler. Testosterone at supraphysiological levels increased the vascular expression of caspase-1 and interleukin-1? (IL-1?), as determined by Western blotting and Elisa, respectively. These data suggest that supraphysiological levels of testosterone induce vascular dysfunction through ROS generation and activation of the NLRP3 inflammasome

A bactéria Legionella pneumophila é um bacilo Gram-negativo, flagelado causador da doença dos legionários e febre de Pontiac. O inflamassoma mais importante no controle da replicação desta bactéria é o composto por Naip5/NLRC4, que é responsável pelo reconhecimento de flagelina. A ativação do inflamassoma de Naip5/NLRC4 pela flagelina induz a ativação de caspase-1, induzindo a formação de poros na membrana, piroptose e controle da replicação desta bactéria. A participação da proteína adaptadora ASC é essencial para a nucleação deste complexo e secreção de citocinas inflamatórias como IL-1? e IL-18 por esta via. Além do controle da replicação de L. pneumophila pelo inflamassoma NLRC4 dependente de caspase-1, foi demonstrado que existe uma via induzida por NLRC4 independente de caspase- 1/11. Dessa forma, camundongos e células Nlrc4-/- são mais susceptíveis à infecção por esta bactéria do que as células Casp1/11-/-. Neste trabalho, nós identificamos que a via independente de caspase-1/11 é composta por Naip5/NLRC4/ASC/Caspase-8 e é essencial para o controle da replicação de Legionella spp. flageladas em macrófagos e in vivo. Através da utilização de BMDMs Casp1/11-/- e Asc/Casp1/11-/- transduzidos com NLRC4-GFP ou ASC-GFP, identificamos que a formação de punctas de NLRC4 e ASC dependem do reconhecimento de flagelina e que ASC é essencial para a formação desses punctas. Também foi identificado que a infecção com L. pneumophila que expressa flagelina leva à ativação de caspase-8 de maneira dependente de ASC e Naip5, mas independente de caspase-1/11. De acordo com esses dados, o silenciamento de caspase-8 em macrófagos Casp1/11-/- aumentou a susceptibilidade dessas células à infecção com L. pneumophila flagelada. Além disso, macrófagos e camundongos Asc/Casp1/11-/- foram tão susceptíveis quanto os Nlrc4- /- e mais susceptíveis que os Casp1/11-/-. Nós observamos que o inflamassoma de NLRC4/ASC/Caspase-8 induz formação de poros e morte celular independente de gasdermina-D (GSDMD). Por meio da utilização de células de camundongos C57BL/6, foi observado que caspase-8 é recrutada para o inflamassoma de Naip5/NLRC4/ASC/Caspase-1. Entretanto, a ativação de caspase-8 só ocorre na 10 ausência de caspase-1 ou GSDMD. Nossos dados sugerem que a ativação de caspase-8 no inflamassoma composto por NLRC4/ASC/Caspase-8 representa uma via alternativa que opera para garantir o controle da replicação de bactérias flageladas em situações nas quais ou caspase-1 ou GSDMD estão inibidas. / Legionella pneumophila is a flagellated Gram-negative bacillus that is the causative agent of the legionnaire\'s disease and Pontiac fever. The most important inflammasome for the control of L. pneumophila replication is the Naip5/NLRC4, responsible for the flagellin recognition. The activation of the Naip5/NLRC4 inflammasome leads to caspase-1 activation, consequently pore formation, pyroptosis and control of bacterial replication. The participation of the adaptor molecule ASC is essential for this complex nucleation and the secretion of inflammatory cytokines like IL-1? and IL-18 by this pathway. Besides the control of L. pneumophila replication by Naip5/NLRC4/Caspase-1 inflammasome, it was demonstrated there are NLRC4 responses independent of caspase-1/11. These explain why mice and macrophages Nlrc4-/- are more susceptible than Casp1/11-/-. In this work, we identified that the caspase-1/11-independent pathway is composed of Naip5/NLRC4/ASC/Caspase-8 and it is essential for the control of flagellated Legionella spp. replication in macrophages and in vivo. Infection of Casp1/11-/- and Asc/Casp1/11-/- macrophages, transduced with NLRC4-GFP or ASC-GFP, showed that flagellin-positive bacteria triggered puncta formation that is ASC-dependent. Accordingly, Naip5 and ASC, but not caspase-1/11, were required for caspase-8 activation in response to flagellated bacteria. Silencing caspase-8 in Casp1/11-/- BMDMs increased the susceptibility to L. pneumophila infection. Furthermore, the macrophages and mice Asc/Casp1/11-/- are as susceptible as Nlrc4-/-, but more susceptible than Casp1/11-/-. We also found that the NLRC4/ASC/Caspase-8 inflammasome induces GSDMD-independent pore formation and cell death. Using C57BL/6 cells, we observed that caspase-8 is recruited to Naip5/NLRC4/ASC/Caspase-1 inflammasome. However, caspase-8 is just activated in the absence of caspase-1 or GSDMD. Our data suggest that caspase-8 activation in the NLRC4/ASC/Caspase-8 inflammasome represents an alternative pathway that operates to ensure the control of flagellated bacteria replication in situations which either caspase-1 or GSDMD are inhibited.

A síndrome do desconforto respiratório agudo (SDRA) e a malária placentária (MP) são complicações da malária, cujos mecanismos imunopatogênicos pouco compreendidos. Neste trabalho, mostramos que camundongos MyD88-/- e Casp1/11-/- não desenvolveu SDRA, morrendo devido ao quadro de anemia severa associada à hiperparasitemia. Posteriormente, demonstrou-se que, embora a patogênese da doença dependa do inflamassoma AIM2, não depende dos inflamassomas NLRP3 e NLRC4 e do eixo IL1. Em uma segunda etapa do projeto foi mostrado que a progressão da PM são decorrentes da ativação das vias de sinalização TLR4/9/MyD88, mas não do TLR2. Ademais, evidenciou-se a participação dos inflamassomas NLRP3 e AIM2, porém não do NLRC4, nesse processo. Por fim, os dados obtidos sugerem que a ativação dessas vias culmina com a liberação de IL-1β que, ao agir em seu receptor, inibe a expressão de transportadores de aminoácidos e glicose, com consequente disfunção do desenvolvimento do feto em camundongos grávidas com MP. Em conclusão, este trabalho apresenta, pela primeira vez, uma associação entre a ativação da via MyD88 e dos inflamassomas pelo plasmódio e a progressão da SDRA e MP. / Acute respiratory distress syndrome (ARDS) and placental malaria (PM) are complications of the malaria, whose the immunopathogenic mechanisms are poorly understood. In that study we showed that MyD88-/- and Casp 1/11-/- mice did not develop ARDS, dying due to severe anemia associated to hyperparasitemia. Subsequently, it was been shown that although such mechanism depends on the AIM2 inflammasome, it does not depend on the NLRP3 and NLRC4 inflammasomes and the IL-1 axis. In a second stage of the project, it should be noted that those complications are due to the activation of the TLR4/9/MyD88 signaling pathways, but not the TLR2. In addition, the participation of the NLRP3 and AIM2 inflammosomes, but not the NLRC4 was shown in that process. Finally, the data suggest that the activation of these pathways culminates with the release of the IL-1β which acts on its receptor inhibiting the amino acid expression and glucose transporters with a consequent dysfunction in the fetal development of pregnant mice with MP. In conclusion, this work makes for the first time an association between the MyD88 pathway activation and inflammasomes by plasmodium and the progression of the ARDS and MP.

Objetivo do estudo foi estudar a participação dos adipócitos e do fígado na inflamação e o papel da microbiota ao longo da progressão da caquexia. Para verificar o comportamento dos adipócitos e do fígado utilizamos ratos Wistar macho de 8 semanas, divididos em dois grupos: i) controle; ii) tumor. Este último foi subdividido em 2 grupos: a) 7º. e b) 14º. dia após a inoculação das células tumorais. Para avaliar o comportamento da microbiota durante o quadro de caquexia utilizamos camundongos C57Bl/6 convencional e germ free de 8-10 semanas, divididos em quatro grupos: i) Convencional controle; ii) Germ Free controle; iii) Convencional tumor; iv) Germ Free tumor. A célula tumoral usada para esse modelo foi Lewis Lung Carcinoma. Adipócitos isolados dos TAB, mesentérico, mais o fígado, mostraram que a via do inflamassoma esta ativa na fase terminal da caquexia. No modelo Germ Free, observamos que a caquexia apresenta-se é acelerada no tecido adiposo epididimal comparado aos camundongos convencionais tumor. Em conclusão, os adipócitos e o fígado desempenha papel importante no estabelecimento da inflamação, enquanto que a simbiose da microbiota parece ser essencial para combater a redução do tecido adiposo. / The goal of this study the role of adipocytes and liver inflammation and the role of microbiota along the progression of cachexia. The main aspects evaluated were increased of the inflammation, alteration in both pathways NF-kB and the inflammasome, and importance of the microbiota during progression of cachexia. To verify the behavior of adipocytes and liver I used Eight weeks-old male rats, I divided into two main groups: i) control; ii) tumor. The latter was divided into 2 groups: a) 7º. and b) 14º. day after tumor cell. To assess the microbial behavior during the development of cachexia I used C57BL/6 conventional mice and Germ Free 8-10 weeks, they were divided into four groups: i) Conventional control; ii) Germ Free control; iii) Conventional tumor; iv) Germ Free tumor. The tumor cell used in this model was Lewis Lung Carcinoma. Adipocytes isolated from TAB, mesenteric further the liver, showed that the inflammasome pathway is active in the terminal phase of cachexia. In model of Germ free mice we observed that cachexia is accelerated in epididymal adipose tissue compared to conventional tumor. In conclusion, adipocytes and liver seem to play a relevant role in the establishment of inflammation, while the microbial symbiosis seems to be essential for combating the reduction of adipose tissue.