L’inflammasome NLRP3 est un complexe multiprotéique responsable notamment de la production d’IL-1β, une cytokine inflammatoire. Les effets délétères de l’inflammasome NLRP3 ont été démontrés dans de nombreuses maladies dont le cancer. Ce travail se concentre sur les effets de NLRP3 dans le contexte du cancer.Dans un premier projet, j’ai étudié l’activation de l’inflammasome NLRP3 dans les MSDC après un traitement par chimiothérapie. Deux chimiothérapies, le 5-Fluorouracile et la Gemcitabine, sont capables d’éliminer de façon spécifique les MDSC, population de cellules immunosuppressives dont la taille augmente en cas de cancer. J’ai découvert que le 5-Fluorouracile et la Gemcitabine activaient l’inflammasome NLRP3 dans les MDSC. En effet, le 5-Fluorouracile et la Gemcitabine provoquent la perméabilisation du lysosome des MDSC, permettant la sortie de la cathepsine B, protéine lysosomale, dans le cytoplasme où elle interagit directement avec NLRP3. Cette interaction active l’inflammasome NLRP3 et la production d’IL-1β. Cette IL-1β est responsable du développement d’une nouvelle population immunosuppressive, les Th17.J’ai ensuite étudié le rôle de NLRP3 dans la différenciation des lymphocytes T CD4 Th2. Dans ces cellules, le rôle de NLRP3 s’effectue indépendamment du reste du complexe multiprotéique qui forme l’inflammasome. Après avoir été induit par la cascade de signalisation de l’IL-2, NLRP3 interagit avec IRF4 (interferon regulatory factor) et agit comme un facteur de transcription sur le promoteur du gène de l’IL-4. L’absence de NLRP3 a pour conséquence une production moins importante d’IL-4 par les Th2 qui sont alors moins fonctionnels / The inflammasome NLRP3 (NOD like receptor pyd containing 3) is a multiprotein complex notably responsible for IL-1β (interleukine-1β) production, an inflammatory cytokine. Negative effects have been observed in various diseases including cancer. My thesis focuses on the effects of NLRP3 in cancer.In my first project, I studied the NLRP3 inflammasome activation in MDSC (myeloïd derived suppressor cells) after a chemotherapy treatment. Two chemotherapies, 5-Fluorouracil and Gemcitabine, are selectively able to kill MDSC, an immunosuppressive population growing during cancer evolution. MDSC’s death restores anti-tumor immunity for a while but another immunosuppressive population is established by MDSC produced IL-1β before their disappearance. I discovered that 5-Fluorouracil and Gemcitabine trigger NLRP3 inflammasome activation in MDSC. 5-Fluorouracil and Gemcitabine induce lysosomal permeabilisation, allowing for Cathepsin B release into the cytoplasm where it directly interacts with NLRP3. That interaction activates the inflammasome and induces IL-1β production which is responsible for the development of another immunosuppressive population, called Th17 cells.I then studied the role of NLRP3 during Th2 differentiation. Here, NLRP3 actions are done independently of the other inflammasome forming proteins. After being induced by IL-2 signalization pathway, NLRP3 interacts with IRF4 (interferon regulatory factor 4) and acts as a transcription factor on the IL-4 promoter gene. Lack of NLRP3 leads to a smaller IL-4 production by Th2 cells which are consequently less functional

L’obésité et le surpoids sont aujourd’hui des problèmes de santé majeurs au niveau mondial touchant près de 1,9 milliard d’adultes. Cet état physiologique se définit comme une accumulation excessive de masse grasse dans l’organisme avec des conséquences néfastes sur la santé. L’obésité tient son origine d’un déséquilibre de la balance énergétique résultant d’une consommation excessive de calories par rapport aux dépenses énergétiques. En accord avec cette idée, les lipides alimentaires représentent les macronutriments les plus caloriques dont la biodisponibilité est assurée par l’intestin. L’accumulation excessive de ces lipides dans les tissus tel que le foie, le tissu adipeux et les vaisseaux sanguins déclenche une inflammation bas-bruit. Cette inflammation bas-bruit est souvent associée à une augmentation modérée de l’endotoxémie métabolique, c’est-à-dire du taux sanguin des lipopolysaccharides bactériens provenant du microbiote intestinal.Dans un premier temps, les travaux de cette thèse avaient pour objectif de définir la contribution de l’intestin dans la mise en place de cette endotoxémie métabolique à travers le rôle du récepteur CD36. En effet, il est montré que ce récepteur participe à la biodisponibilité des lipides en favorisant leur absorption et la formation des chylomicrons qui véhiculent les lipopolysaccharides. Les données obtenues ont permis de montrer que CD36 participe aux situations d’endotoxémie métabolique en régulant l’écologie microbienne du site d’absorption des lipides alimentaires.Dans un second temps, ces travaux se sont intéressés à la contribution de la réponse inflammatoire dans les mécanismes d’absorption des lipides alimentaires au travers du rôle de l’inflammasome NLRP3. Ils ont permis de montrer que cet inflammasome participe à la digestion et à l’absorption des lipides alimentaires lors d’un régime hyperlipidique obésogène.L’ensemble de ces données permet d’établir le rôle majeur de l’intestin dans la mise en place de l’endotoxémie et des processus inflammatoires conduisant à une situation d’obésité. De plus, le récepteur CD36 et l’inflammasome NLRP3 pourrait constituer des cibles thérapeutiques de choix pour limiter la mise en place de cet état physiologique. / Nowadays, obesity and overweight are major health problems affecting nearly 1.9 billion adults. This physiological state is defined as an excessive accumulation of fat mass in the body with harmful consequences on health. Obesity is caused by a disequilibrium in the energy balance resulting from an excessive calorie consumption in relation to the energy expenditure. According to this concept, dietary fats represent the most caloric macronutrients whose bioavailability is ensured by the intestine. Excessive accumulation of these lipids in tissues such as liver, adipose tissue and blood vessels triggers low-grade inflammation. This low-grade inflammation is often associated with a moderate increase in metabolic endotoxemia, i.e. an increase in the blood level of bacterial lipopolysaccharides coming from the intestinal microbiota.First, the aim of this thesis was to elucidate the contribution of the intestine to the development of the metabolic endotoxemia through the role of the CD36 receptor. Indeed, this receptor participates in the bioavailability of dietary lipids by promoting their absorption and the formation of chylomicrons able to carry the lipopolysaccharides. The data obtained showed that CD36 could participates in metabolic endotoxemia situations by regulating the microbial ecology of the food lipid absorption site.In a second step, this work focused on the contribution of the inflammatory response to the absorption mechanism of dietary lipids through the role of the NLRP3 inflammasome. It showed that this inflammasome is involved in the digestion and absorption of dietary lipids in high-fat diet-induced obesity.The data obtained allow to establish the major role of the intestine in the development of endotoxemia and inflammation during the development of obesity. In addition, the CD36 receptor and the NLRP3 inflammasome may be therapeutic targets to limit the development of this physiological condition.

Pattern recognition receptors coordinate innate immune responses by sensing infection or injury. Nucleotide-binding, leucine rich repeat, and pyrin domain-containing protein 3 (NLRP3) is a cytosolic PRR which perceives diverse pathogenic and sterile insults. NLRP3 orchestrates inflammatory signaling responses by forming inflammasomes with the adaptor protein apoptosis-associated speck like protein with a caspase recruitment domain (ASC) and the cysteine protease caspase-1. Assembly of the intracellular macromolecular inflammasome complex culminates in proximity-induced autocatalysis of caspase-1. Caspase-1 activation promotes cell death by pyroptosis and activation and secretion of proinflammatory cytokines interleukin (IL)-1β and IL-18. While NLRP3-mediated inflammation protects against bacterial, fungal, viral, and parasitic infections, aberrant NLRP3 activation is implicated in numerous inflammatory diseases and heritable syndromes. Mechanistically, inflammasome activation requires a preliminary NF-κB-activating priming step (signal 1) and a subsequent NLRP3-specific stimulus (signal 2). While there is enormous molecular diversity among NLRP3-specific agonists, this second signal appears to engage a common pathway involving cation flux. Furthermore, NLRP3 associates with mitochondria and mitochondrial damage is implicated in NLRP3 activation, although the precise role for mitochondria in inflammasome assembly remains controversial. We previously demonstrated that the mitochondrial phospholipid cardiolipin binds to NLRP3 and is critical for NLRP3 inflammasome activation. Here, we further investigated how mitochondria contribute to NLRP3 activation. We found that liposomes containing molar concentrations of cardiolipin that resemble mitochondrial cardiolipin levels can induce NLRP3-dependent caspase-1 autoactivation. Unexpectedly, we discovered that caspase-1 binds directly to cardiolipin, causing inflammasome-independent caspase-1 complex formation and autocatalysis at higher cardiolipin densities. Finding that caspase-1 and NLRP3 are independently capable of binding to cardiolipin, we more thoroughly examined the association of inflammasome components with mitochondria. Normally confined within mitochondrial inner membranes, cardiolipin relocates to outer membranes of stressed mitochondria. We found that reactive oxygen species (ROS) produced in response to signal 1 facilitate cardiolipin externalization to the outer membrane during priming. We also determined that this coincides with ROS-dependent recruitment of NLRP3 and caspase-1 to the outer membrane of mitochondria at priming. In contrast, we found that NLRP3 activation by the signal 2 agonist nigericin induces calcium-dependent recruitment of the adaptor ASC to mitochondria and caspase-1 activation. Finally, to determine what type of mitochondrial damage was necessary to promote NLRP3 inflammasome activation, we examined how different NLRP3 agonists affect mitochondria. We found substantial variability in the extent of mitochondrial damage induced among different NLRP3 agonists. Collectively, our findings illustrate that mitochondria serve as innate immune signaling platforms through multiple stages of NLRP3 inflammasome activation. Further, paralleling lipid A interactions with caspase-11, we have demonstrated that caspase-1 is capable of binding to the phospholipid cardiolipin.

Age-related macular degeneration (AMD) is a leading cause of visual impairment and blindness, affecting over 30 million people worldwide. It is characterized by the appearance of insoluble deposits known as drusen in the outer retina, between the retinal pigment epithelium (RPE) and Bruch's membrane. Drusen are heterogeneously composed of many compounds, including cholesterol, amyloid-β, and complement proteins. AMD also involves the accumulation of pigments collectively termed lipofuscin in RPE lysosomes. The underlying causes of AMD are unknown, but studies have implicated inflammatory processes in its pathogenesis.

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.

京都大学 / 0048 / 新制・課程博士 / 博士(医学) / 甲第21626号 / 医博第4432号 / 新制||医||1033(附属図書館) / 京都大学大学院医学研究科医学専攻 / (主査)教授 高橋 淳, 教授 宮本 享, 教授 林 康紀 / 学位規則第4条第1項該当 / Doctor of Medical Science / Kyoto University / DFAM

Recently discovered methods for both diagnosis and treatment management of heart failure (HF) and other cardiovascular diseases include the use of molecular imaging modalities such as positron emission tomography (PET). As such, PET radiotracers have been developed and show strong evidence for quantifiable sympathetic nervous system (SNS) imaging in animals and humans using radioligands that target the norepinephrine transporter (NET). In this study, the cardiac sympathetic nervous system imaging probe [¹⁸F]meta-fluorobenzylguanidine ([¹⁸F]mFBG) was evaluated in Sprague Dawley (SD) rats and hypothesized to i) have measurable NET-dependent uptake kinetics; ii) possess measurable NET reuptake, iii) be stored into vesicles, and remain metabolically stable within the myocardium, iv) be sensitive to changes in sympathetic tone, and v) discriminate between healthy and diseased animals. Using the selective NET inhibitor desipramine (DMI) and nonselective extraneural and neural uptake inhibitor phenoxybenzamine (PBZ) we observed 30-35% and ~70% reduced uptake in the myocardium, respectively. Neuronal ablation with 6-hydroxydopamine (6-OHDA) resulted in a 36% loss of myocardial retention. DMI and PBZ chase dosing revealed no change in myocardial washout by PET, suggesting minimal reuptake of the tracer and preferential clearance into blood. Pretreatment with the vesicular monoamine transporter (VMAT) inhibitor reserpine (RSP) reduced myocardial retention by 34% within 5 minutes in comparison to baseline, providing evidence for intraneural vesicular retention. Dahl salt-sensitive (DSS) rats were induced with HF via high salt (HS, 8%) diet. After 16 weeks, rats kept on HS diet showed ~32% reduced myocardial uptake in comparison to low salt (LS) controls. Non-invasive PET imaging of HF is therefore sensitive to the expected changes in myocardial uptake in small animal imaging. The myocardial cholinergic system was evaluated using the vesicular acetylcholine transporter (VAChT) ligand [¹⁸F]fluoroethoxybenzylvesamicol ([¹⁸F]FEOBV) in CD-1 mice. We hypothesized that i) [¹⁸F]FEOBV uptake in the myocardium is VAChT dependent; and more specific in the absence of isoflurane anesthesia. Baseline uptake was observed in the ventricles. However, pretreatment with vesamicol in the presence and absence of isoflurane did not reduce myocardial activity. Analysis of PET images in mice with differential cardiac VAChT expression showed minimal changes in blood and cardiac activity. These studies have demonstrated a lack of specific binding of [¹⁸F]FEOBV in the myocardium of mice, rendering this imaging probe unfit in interpreting cholinergic function in small animals. The second half of the thesis is focused on the development of a radiolabeling technique using novel iminophosphorane precursors, and their subsequent reactivity and application using the naturally abundant (99% carbon-12) and positron emission tomography (PET) imaging isotope (carbon-11). We hypothesized that the reaction of iminophosphorane precursors with [¹¹C]CO₂ with allow us to i) synthesize a myriad of labelled isocyanate derived functional groups ii) apply this chemistry to label relevant radiopharmaceuticals in high yield and molar activity. Optimization of reaction conditions was performed, and a substrate scope was developed. Using the naturally abundant carbon isotope, we synthesized isocyanate derived functional groups such as carbamates, thiocarbamates, ureas, and amides in 63-94% yield. Pharmaceuticals such as regorafenib, URB694, and melatonin were synthesized in 60-72% yield. When applied to carbon-11 radiochemistry, labelled products were produced in 32-84% radiochemical yield (RCY). Radiopharmaceuticals such as [¹¹C]URB694 and [¹¹C]glibenclamide were synthesized in high yield and molar activity suitable for preclinical evaluation. We have demonstrated the utility of iminophosphorane precursors in synthesizing labelled functional groups and relevant radiopharmaceuticals in high yields, enabling their use for future preclinical or clinical studies. The recent development of the potent and selective nod-like receptor protein-3 (NLRP3) inhibitor MCC950 has demonstrated remarkable application as a therapeutic in reducing macrophage infiltration and aortic lesion area, but has yet to be applied to PET imaging due to poor synthesis yields. We hypothesized that i) using our previously established iminophosphorane chemistry we can synthesize [¹¹C]MCC950, and ii) selective uptake of [¹¹C]MCC950 occurs in aortic atherosclerotic lesions. We successfully radiolabeled [¹¹C]MCC950 in 45 ± 4 % RCY (27 ± 2 GBq/µmol). Plasma metabolite analysis revealed 94% intact tracer after 15 minutes, and ex vivo autoradiography on excised aortas showed heterogeneous uptake in atherosclerotic plaques of ApoE⁻ᐟ⁻ mice. Pretreatment with nonradioactive MCC950 resulted in significantly increased uptake in aortic lesions (48 ± 17 %ID/m² vs 104 ± 15 %ID/m²), without significantly increasing plasma free fraction (1.3 ± 0.4% vs 1.7 ± 0.8%). The data suggests increased specific binding following blockade which may be due to biochemical mechanisms such as disaggregation of NLRP3 oligomers, artificially increasing the available number of binding sites. Thus, the data suggest [¹¹C]MCC950 uptake demonstrates specific binding and may therefore prove useful as an in vivo imaging probe to detect NLRP3-mediated inflammation in atherosclerosis.

Diese Dissertation (Monographie) wurde in Abteilung für Kardiologie des Uniklinikums Leipzig durchgeführt. Zusammenfassend zeigen die Experimente, dass extrazelluläre NLRP3 Oligomere von Makrophagen internalisiert werden und eine pro-inflammatorische und chemoattraktive Wirkung besitzen.

Traumatic brain injury (TBI) is a major medical concern that has demonstrated to be particularly challenging to treat because of the disparity amongst injury modes and severities. Increased use of explosive devices during combat has caused blast TBI (bTBI) to become a widespread consequence in military and Veteran populations, and impact-related trauma from contact-related sports or motor vehicle accidents has made mild impact-induced TBIs (concussion) a major health problem. There is a high risk for those who have sustained a TBI to develop behavioral and cognitive disorders following injury, and these symptoms can present as delayed onset, causing diagnosis to be a major feat when planning for treatment and long-term healthcare. Both preclinical and clinical studies report the neuropathological changes following TBI, yet investigating the distinct mechanistic changes in blast and impact trauma that contribute to pathological disparities has yet to be elucidated. Microglia dynamics play a key role in initiating the inflammatory response after injury, as microglia become activated by undergoing morphological changes that influence their function in the injured brain, and unique signaling pathways influence their functional inflammatory states. While previous literature report on the unique responses of microglia, their mediated-inflammatory responses are still not well defined. This work aimed to investigate the acute and subacute responses of microglia to injury through their diverse activation states following blast and impact trauma. The work herein employed rodent models to investigate these changes, finding that microglia activation was spatially and temporally heterogeneous within and across injury paradigms. Three days following bTBI, activated microglia in the cortex displayed morphologies similar to microglia that are known to increase their interactions with dysfunctional synapses, while dystrophic microglia were prevalent in the hippocampus seven days following injury. Moreover, transhemispheric changes in microglia activation were noted following impact TBI, with stressed/primed microglia responding to immune challenges of the cortex at three days, whereas a unique morphological state that was markedly different from those traditionally reported in CNS injury and disease was present within the hippocampus three- and seven-days following injury. State-of-the-art cell sorting techniques were used for in vivo analysis of microglia, which also exhibited that functional changes of microglia vary between injury paradigms, providing insight into how differences in primary insult may elicit distinct signaling pathways involved in microglia-mediated inflammatory responses. These in vivo studies were then crucial in understanding the malleable responses of microglia to complex injuries such as "blast plus impact" TBI, indicating that phenotypic changes in microglia following this injury are also unique and spatially heterogeneous. To date, therapeutic efforts for TBI are limited due to the lack of understanding the underlying mechanisms that influence TBI pathology. This work also investigated novel therapeutic targets, noting that administration of polyester nanoparticles restored microglia to baseline levels following impact. The fundamental research presented in this study is innovative and advantageous as it can provide essential data into targeted and personalized treatments that can improve long-term healthcare and ultimately, the quality of life for those suffering from a TBI. / Doctor of Philosophy / Traumatic brain injury (TBI) is a major medical concern that has demonstrated to be particularly challenging to treat because of the differences in injury modes and severities. Increased use of explosive devices during combat has caused blast TBI (bTBI) to become a widespread result in military and Veteran populations, and impact-related trauma from contact sports or motor vehicle accidents has made mild impact-induced TBIs (concussion) a major health problem. There is a high risk for those who have sustained a TBI to develop behavioral and cognitive disorders following injury, and these symptoms can present later on, causing diagnosis to be a major feat when planning for treatment and long-term healthcare. Microglia play a key role in inducing the inflammatory response after injury, as they change shape and size, which then influences their function in the injured brain. Although prior research reports on the unique responses of microglia, their effects on inflammation following TBI are still not well defined. This work aimed to investigate the early responses of microglia to injury through their diverse activation states following blast and impact trauma. The experiments in this study used animal models, finding that microglia activation can be distinct across time and brain regions, which may be injury-type-specific. To date, therapeutic efforts of TBI are limited due to the lack of understanding the underlying mechanisms that influence TBI pathology. This work also investigated beneficial treatments for TBI, noting that administration of nanoparticles helped restore microglia to levels similar to the control group. The fundamental research presented in this study is innovative and important as it can provide essential data into targeted and personalized treatments that can improve long-term healthcare and ultimately the quality of life for those suffering from a TBI.

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.