Activation of the NLRP3 inflammasome has been shown in response to numerous activators; here we show that the oxazolidinone antibiotic linezolid results in both the NLRP3-dependent in vitro release of the proinflammatory cytokine IL-1 Α; and in vivo neutrophilic influx following its intraperitoneal administration. Clinical use of linezolid is commonly limited by hematologic side effects; herein we also show NLRP3-deficiency protected animals against linezolid-induced effects on the bone marrow. Importantly, all previously described activators of the NLRP3 inflammasome have required the generation of reactive oxygen species (ROS). Linezolid is however unique amongst NLRP3 agonists in that its ability to activate the NLRP3 inflammasome in a ROS-independent manner. The pathways for ROS-dependent and ROS-independent NLRP3 activation converge upon mitochondrial dysfunction and specifically the mitochondrial lipid cardiolipin. We demonstrated that interference with cardiolipin synthesis specifically inhibits NLRP3 inflammasome activation. These findings firstly suggests that ROS generation is not the canonical activator of NLRP3 but rather an intermediary step leading to the mitochondrial perturbation that is tied to NLRP3 inflammasome activation and also implicate the involvement of mitochondrial lipid cardiolipin in this process; secondarily, linezolid-induced NLRP3 activation may account for thetoxicity associated with prolonged usage of this antibiotic.

L’asthme est une maladie inflammatoire chronique des voies aériennes. Chez les individus sensibles, l’inhalation d’allergènes entraine une inflammation pulmonaire se traduisant par des épisodes récurrents de toux, de difficultés respiratoires et une sécrétion de mucus. Des études réalisées chez l’animal ont mis en évidence un rôle crucial des lymphocytes Th2 et des cytokines associées (IL-4, IL-5 et IL-13). Plus récemment, il a été montré que les lymphocytes Th17 participaient à la physiopathologie de l’asthme. La présente étude s’intéresse à une cytokine majoritairement produite par les Th17 : l’IL-22. Différents travaux indiquent que cette cytokine serait impliquée dans l’immunitémucosale où elle exercerait des effets protecteurs ou pro-inflammatoires en fonction du modèle expérimental étudié. En utilisant un modèle murin d’inflammation pulmonaire allergique induite par l’ovalbumine, nous avons montré que l’IL-22 jouait un rôle pro-inflammatoire au cours de l’induction de l’asthme allergique puisque les souris déficientes en IL-22 développent une forme atténuée de la maladie. A l’inverse, nous avons constaté que l’IL-22 avait un effet protecteur dans la phase effectrice, et que cet effet était dépendant de l’IL-17A. Nos travaux mettent donc en lumière une double fonction de l’IL-22 dans l’asthme allergique chez la souris. En parallèle de ce travail, nous nous sommes intéressés au rôle de l’IL-1 et de l’inflammasome NLRP3 dans ce même modèle d’inflammation pulmonaire. Enfin, une troisième étude a permis de mettre en lumière un rôle encore inconnu de l’interleukine-33 dans l’activation des cellules dendritiques au cours de la mise en place de la réponse asthmatique. / Asthma is a heterogenous inflammatory disorder of the airways characterized by chronic airway inflammation, airway hyper-reactivity and by symptoms of recurrent wheezing, coughing and shortness breath. Understanding of the role of allergy and Th2 cells in asthma has benefited from mouse model of allergic asthma. Recently, several studies highlighted Th17 involvement in asthma pathogenesis. In the present study, we investigate the role of IL-22, a Th17-related cytokine, in a mouse model of allergic lung inflammation induced by ovalbumin. First, using IL-22 deficient mice, we demonstrated a pro-inflammatory role of IL-22 during the sensitization phase. In contrast, we observed a protective function of IL-22 during the effective phase. This protective effect of IL-22 seems to be dependent of IL-17. In conclusion, we demonstrate here a dual role of IL-22 in asthma pathogenesis. Since interleukin-1_ is critical for Th17 polarization in human, we also investigated the role of IL-1 signalling and NLRP3 inflammasome in our model of allergic airway inflammation. We showed that NLRP3 inflammasome and IL-1R/IL-1 pathway are critical to induce allergic lung inflammation, even in the absence of adjuvant. Finally, we studied the effect of interleukin-33 on dendritic cells activation and Th2 priming during antigen sensitization and in established asthma.

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

The innate immune system are a cooperation of many components – receptors being one of them. Both membrane-bound and cytosolic receptors play a large role in the defence system against pathogens and danger. NLRP3 is a receptor which assembles a protein complex called inflammasome in response to cytosolic stress and is responsible for many autoimmune diseases if it malfunctions. The activation of the NLRP3 inflammasome leads to secretion of inflammatory cytokines and in many cases to programmed cell death. The structure, function and activation of the NLRP3 inflammasome is still not fully understood and the urge to understand the mechanisms behind are important for future medical improvements. The aim was to anchor the NLRP3 inflammasome by the cell membrane - By Overlap PCR, the NLRP3 cDNA was fused extracellular and trans-membrane parts of the TLR4 cDNA to anchor the NLRP3 to the membrane and in turn analyse the inflammasome with LPI™ technology. Multiple primers and a TLR4 nucleotide were designed and the NLRP3 was amplified with specific overhangs by PCR. The fusion protein was successfully linked together by Overlap PCR but not confirmed by sequencing. The gene fusion demands high quality primers for amplification and further evaluation must be made to the details of the laboratory. To anchor the protein complex to the cell membrane, continue to be of full importance and can be an asset in many structural studies and biopharmaceuticals trials.

Age-related macular degeneration (AMD) is a principal cause of blindness in the United States and other industrialized nations. An estimated 10 million Americans are afflicted with AMD, which is comparable in scope to the 12 million living with cancer, or the 5 million with Alzheimer’s disease. The prevalence of AMD steadily increases with age, affecting 2% of the population at age 40, and one in four people by age 80. For reasons that are not fully understood, AMD is more common in lightly-pigmented and female populations. Treatment of AMD is largely an unmet need: There are no FDA approved therapies except for a small percentage of individuals with end-stage disease. This dissertation investigates the mechanisms of AMD pathogenesis and offers insight into novel therapeutic strategies for this disease.

Wear particles and ions from cobalt-chromium-molybdenum (CoCrMo)-based implants have been shown to cause adverse immune responses, including periprosthetic osteolysis leading to aseptic loosening, the main cause of implant failure. Previous studies have shown that these wear and corrosion products can lead to the release of inflammatory cytokines, including interleukin-1β (IL-1β), suggesting the involvement of the NLRP3 inflammasome. However, the mechanisms leading to IL-1β release have not been fully elucidated. The primary objectives of this thesis were to determine if, in murine macrophages, IL-1β release induced by micrometre-size CoCrMo particles and nanometre-size chromium oxide (Cr2O3) particles is: 1. Caspase-1-dependent; 2. Reduction-oxidation (redox)-dependent; and 3. NLRP3 inflammasome-dependent. Additionally, the effects of metal ions (Co2+, Cr3+, and Ni2+) on NLRP3 inflammasome activation and the effects of matrix metalloproteinase (MMP) inhibition on IL-1β release induced by CoCrMo particles were analyzed. Results showed that IL-1β release induced by CoCrMo particles was partly caspase-1-, redox-, and MMP-dependent, but NLRP3 inflammasome-independent. On the other hand, IL-1β release induced by Cr2O3 particles appeared to be NLRP3 inflammasome-dependent. Finally, IL-1β release induced by Cr3+, but not Co2+, appeared to be NLRP3 inflammasome-dependent, while Ni2+-induced IL-1β release appeared to be only partially NLRP3 inflammasome-dependent, suggesting that other pathways may also be involved. These findings, which provide additional insights into the mechanisms leading to IL-1β release induced by wear particles and ions from CoCrMo-based implants, may help the future development of therapeutic treatments to modulate wear product-induced inflammation and increase implant longevity.

Inflammasomes are cytosolic protein platforms whose assembly/activation, in order to resolve various types of threats, can be triggered by a multitude of microbial and host derived stimuli. Their discovery was the key breakthrough in the understanding of inflammation initiation. Although beneficial in clearing microbes and restoring physiological or tissue homeostasis, aberrant or excessive stimulation of inflammasomes leads to various pathologies. The activation of NLRP3 inflammasome is associated with the physio-pathogenesis or the potentiation of the severity of so many diseases including respiratory diseases, such as asthma, COPD, COVID-19, malaria, diabetes, atherosclerosis, etc. Considering the extent of the NLRP3 inflammasome in diseases, therapies targeting the inhibition of its activation appear as a promising approach for the management of a wide range of diseases. The objective of this research work was to investigate the potential of Rwandan medicinal plants in the inhibition of the NLRP3 inflammasome. To this end, we first carried out a field survey to collect ethnobotanical information on plants used for the treatment of asthma in Rwanda. The present thesis is divided into two main parts:- The ethnobotanical study and screening of Rwandan medicinal plants with inhibitory activity on NLRP3 inflammasome activation;- The isolation and structure characterization of the active ingredients responsible for NLRP3 inflammasome inhibition, and the investigation of antiplasmodial activity of selected plants.In the first part, 19 herbal recipes were reconstituted from 61 medicinal plants based on an ethnobotanical survey. Most of these recipes exhibited significant inhibition of the NLRP3 inflammasome activation. The four recipe extracts named R03Cn and R07Kn aqueous extracts, and R10MK and R19Sz organic extracts significantly downregulated the activation of caspase-1 (more than 70% at 50 µg/ml). As R19Sz revealed the best NLRP3 inflammasome inhibitory activity and was less toxic it was hence selected for further investigations. In the second part of the work, two unstudied plants from the R19Sz recipe were submitted to a bio-guided fractionation. This led to the isolation and identification of compounds including caffeic acid, isoquercetin, kaemferol-3-O-galactoside as being the major constituents responsible for the inhibition of NLRP3 inflammasome activation in H. noldeae, and to the isolation and identification of 7-O-glucopyranosyl-isoprunetin, isoprunetin, and luteolin as the main compounds responsible for the inhibitory activity of NLRP3 inflammasome activation in E. montanum. Additionally, in vitro anti-plasmodial activities coupled to cytotoxicity assays of some bio-guided fractionation of E. montanum led to the isolation and determined antiplasmodial activity of Eucomic acid (IC50 = 0.057µg/ml), 7-O-glucopyranosyl-isoprunetin (IC50 = 0.113 µg/ml), isoprunetin (IC50 = 0.042 µg/ml), Isoluteolin (IC50: 0.121 µg/ml) as the main compounds responsible for the antiplasmodial activity with the high selectivity indexes of 6 577, 2646.7; 5264.3 and 2005.8, respectively .Overall, the present work has potentiated the role of Rwandan herbal medicines in the treatment of asthma and malaria while paving ways at the molecular and functional levels allowing to say that these plants likely act on inflammation by inhibiting the NLRP3 inflammasome activation. / Option Biologie moléculaire du Doctorat en Sciences / info:eu-repo/semantics/nonPublished

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