Hintergrund und Fragestellung: Das NLRP3-Inflammasom ist Teil des angeborenen Immunsystems und spielt eine wichtige Rolle für pro-inflammatorische Prozesse im Rahmen der sterilen Entzündung und der Pathogenese der Atherosklerose. Das NLRP3-Inflammasom wird nicht nur durch molekulare Strukturen von Krankheitserregern, sondern auch durch endogene Gefahrensignale, wie beispielsweise oxidiertes LDL, Cholesterinkristalle, oxidativer Stress oder extrazelluläres ATP, aktiviert. Bei Aktivierung bilden sich hochmolekulare NLRP3-Inflammasomkomplexe, bestehend aus dem NLRP3-Rezeptor, dem Adapterprotein ASC und dem vom Adapterprotein gebundenen Effektorenzym Caspase-1. Im Zuge der Inflammasom-Aktivierung kommt es zur Induktion von Pyroptose, einem programmierten und mit Entzündung assoziierten Zelltod, der mit einer Porenbildung in der Zellmembran einhergeht. Dabei werden pro-inflammatorische Zytokine in ihre bioaktiven Formen gespalten und in den extrazellulären Raum freigesetzt. Neben den Zytokinen können auch ganze Inflammasomkomplexe freigesetzt werden, wodurch diese ebenfalls in die Blutzirkulation gelangen. Die Bedeutung und Wirkung dieser extrazellulären NLRP3-Inflammasome ist noch weitgehend unbekannt. Daher wurde in den beiden wesentlichen Fragestellungen der Arbeit untersucht, ob Endothelzellen extrazelluläre Inflammasomkomplexe internalisieren und dadurch pro-atherogene Effekte ausgelöst werden können und ob zirkulierende Inflammasomkomplexe im menschlichen Blut nachgewiesen werden können. Methoden und Ergebnisse: Extrazelluläre Inflammasomkomplexe konnten aus Zellüberstand von mit Inflammasom-Aktivatoren behandelten humanen Monozyten isoliert werden. Mittels Western-Blot erfolgte der Nachweis des Adapterproteins ASC und des NLRP3-Rezeptors. Durch den NLRP3-Inhibitor MCC950 konnte eine Reduktion der Freisetzung von Inflammasomkomplexen in den Zellüberstand gezeigt werden. Für die Untersuchungen der Wirkung extrazellulärer Inflammasomkomplexe auf Endothelzellen wurde eine Methode zur Isolation von fluoreszenzmarkierten NLRP3-YFP-Inflammasomkomplexen aus einer stabilen NLRP3 (p.D303N)-YFP HEK-Zelllinie etabliert. Histologische Analysen zeigten, dass diese von humanen koronaren Endothelzellen aufgenommen werden können. Die immunhistochemischen Befunde bestätigten sich in der Durchflusszytometrie (Image StreamR). Die Auswertung der immunfluoreszenzmikroskopischen Aufnahmen ergab, dass im Mittel 9 % ± 2,3 % der humanen umbilikalvenösen Endothelzellen und 26 % ± 9,6 % der humanen koronararteriellen Endothelzellen NLRP3-YFP-Inflammasomkomplexe internalisierten. Expressionsanalysen zeigten, dass durch die Behandlung von humanen Endothelzellen mit NLRP3-Inflammasomkomplexen eine signifikant gesteigerte Produktion des Oberflächenadhäsionsmoleküls ICAM1 induziert wird. Im Vergleich zu unbehandelten Zellen waren die mRNA-Expression 2,5-fach und die Proteinlevel 6,9-fach erhöht. Die funktionelle Relevanz wurde durch den Nachweis einer vermehrten Adhäsion von Monozyten deutlich. Mit Calcein gefärbte Monozyten wurden vier Stunden mit koronararteriellen Endothelzellen inkubiert. Die anschließend photometrisch erhobenen Daten zeigten eine um 49,6% verstärkte Adhäsion von Monozyten an die vorher mit extrazellulären Inflammasomkomplexen behandelten Endothelzellen im Vergleich zu unbehandelten Zellen. Die Monozytenadhäsion an das Endothel stellt einen essentiellen Schritt in der Pathogenese der Atherosklerose dar. Diese Zellkultur-Ergebnisse zeigen, dass das extrazelluläre NLRP3-Inflammasom relevanten Einfluss auf das Endothel nehmen kann. Um die klinische Relevanz von extrazellulären NLRP3-Inflammasomen zu eruieren, wurde humanes Serum auf das Adapterprotein ASC hin untersucht. Im Rahmen dieser Dissertationsarbeit wurde die Messung von ASC bei Patienten mit Sepsis als Positivkontrolle im Vergleich zu gesunden Probanden als Negativkontrolle etabliert und validiert. Im Western-Blot konnte gezeigt werden, dass Patienten mit Sepsis mehr ASC-Komplexe aufweisen als gesunde Probanden. Dieser Befund konnte in der quantitativen Auswertung mittels ASC-ELISA bestätigt werden. Es waren signifikant höhere ASC-Konzentrationen in den Serumproben der Patienten mit Sepsis (3,2 ± 0,7 ng/ml) als in gesunden Kontrollen (0,3 ± 0,1 ng/ml) nachweisbar. Diskussion: Diese Ergebnisse zeigen, dass extrazelluläre NLRP3-Inflammasomkomplexe von humanen koronararteriellen Endothelzellen aufgenommen werden und dort pro-atherosklerotische Prozesse induzieren. Die erhobenen Daten weisen damit auf eine neue Domäne der zellulären Signaltransduktion hin, welche für Endothelzellen und im Rahmen der sterilen Entzündung relevant scheint. Die Messung erhöhter ASC-Spiegel im Serum von Sepsis-Patienten lässt ebenfalls eine Assoziation von Inflammasomkomplexen mit im Körper ablaufender systemischer Inflammation annehmen. Auf dem Boden der Befunde müssen weitere Studien beantworten, ob sich die Quantifizierung zirkulierender Inflammasomkomplexe als Biomarker oder zur Risikostratifizierung eignet. Des Weiteren unterstützen die Daten zukünftige Ansätze einer therapeutischen Modulation des intra- und des extrazellulären NLRP3-Inflammasoms.

京都大学 / 新制・論文博士 / 博士(農学) / 乙第13497号 / 論農博第2901号 / 新制||農||1093(附属図書館) / 学位論文||R4||N5403(農学部図書室) / (主査)教授 谷 史人, 教授 佐々木 努, 教授 保川 清 / 学位規則第4条第2項該当 / Doctor of Agricultural Science / Kyoto University / DFAM

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A esclerose múltipla (EM), doença neurodegenerativa do sistema nervoso central (SNC) com característica auto-imune e inflamatória, com eventos iniciais, bem como a evolução da EM. É uma doença heterogênea (três principais formas clínicas) e multifatoriais. A imunidade inata demonstrou recentemente ser um fator importante na EM e as variantes genéticas dos componentes do inflamassoma têm sido associadas a doenças autoimunes e neurodegenerativas, com isso hipotetizamos que o inflamassoma e suas citocinas IL-1Beta e IL-18, podem representar importantes contribuintes na patogênese da EM e eventualmente explicar, pelo menos em parte, a heterogeneidade observada em pacientes com EM. Fizemos uma análise multivariada que foi realizada com base na forma clínica (recorrente remitente/RR, primária progressivo /PP ou secundário progressiva /SP, índice de gravidade (EDSS) e índice de progressão (IP). Os monócitos do sangue periférico (PBMC) dos pacientes foram examinados para ativação do inflamassoma (Produção de IL-1Beta e IL-18, clivagem de caspase-1). Com os objetivos de avaliar a contribuição do inflamassoma na EM, em termos de (a) efeito genético sobre o desenvolvimento, gravidade e / ou prognóstico, e (b) ativação complexa de células de sangue periférico como uma forma de avaliar a inflamação sistêmica. Para isso, utilizamos variantes genéticas funcionais em componentes do inflamassoma, que foram analisadas em uma coorte de pacientes com EM, pelo uso de ensaios específicos de alelos e qPCR. A analise multivariada resultou em associação com a variante -511C / T IL1B ganho de função, sendo essa mais frequente em formas progressivas (especialmente SP) do que em RR. A variante de ganho de função NLRP3 Q705K resultou mais frequente em pacientes com EDSS > 3 do que em pacientes com EDSS < 3 e, consequentemente, esse SNP está associado a um IP mais elevado. A análise de PBMC mostrou que as células de indivíduos EM, são mais propensas a responder a um estímulo NLRP3 clássico (isto é, LPS) do que as dos doadores saudáveis. Em conjunto, esses achados indicaram que os pacientes com EM apresentam uma desregulação no inflamassoma NLRP3, podendo ser avaliada no sangue periférico facilitando um prognóstico, e que esse perfil pode ser secundário a um mecanismo genético pró-inflamassoma / The multiple sclerosis (MS), neurodegenerative disease of the central nervous system (CNS) with autoimmune and inflammatory characteristics, with initial events, as well as the evolution of MS, are heterogeneous (three main clinical forms) and multifactorial. Innate immunity has recently been shown to be an important factor in MS and the genetic variants of the components of inflammassoma have been associated with autoimmune and neurodegenerative diseases, thereby hypothesizing that the inflammassoma and its IL-1Beta and IL-18 cytokines may represent important contributors in the pathogenesis of MS and possibly explain, at least in part, the heterogeneity observed in MS patients. We performed a multivariate analysis that was performed based on clinical form (recurrent recurrent / RR, progressive primary / PP or progressive secondary / SP, EDSS and progression index.) Peripheral blood mononuclear cells (PBMC) of patients were examined for inflammatory activation (IL-1Beta and IL-18 production, caspase-1 cleavage). With the objectives of evaluating the contribution of inflammassoma in MS in terms of (a) genetic effect on development, severity and / or prognosis, and (b) complex activation of peripheral blood cells as a way of assessing systemic inflammation. For this, we used functional genetic variants in components of the inflammassoma, which were analyzed in a cohort of MS patients, through the use of specific allele and qPCR assays. For this, we used functional genetic variants in components of the inflammassoma, which were analyzed in a cohort of MS patients, through the use of specific allele and qPCR assays. Multivariate analysis resulted in association with the -511C / T IL1B function gain, which is more frequent in progressive forms (especially SP) than in RR. The gain variant of NLRP3 Q705K function was more frequent in patients with EDSS > 3 than in patients with EDSS < 3 and, consequently, this SNP is associated with a higher PI. PBMC analysis showed that cells from MS individuals are more likely to respond to a classical NLRP3 (ie LPS) stimulus than healthy donors. Taken together, these findings indicated that patients with MS have a dysregulation in the NLRP3 inflammassoma and can be evaluated in the peripheral blood facilitating a prognosis and that this profile may be secondary to a pro-inflammatory genetic mechanism

A esclerose múltipla (EM), doença neurodegenerativa do sistema nervoso central (SNC) com característica auto-imune e inflamatória, com eventos iniciais, bem como a evolução da EM. É uma doença heterogênea (três principais formas clínicas) e multifatoriais. A imunidade inata demonstrou recentemente ser um fator importante na EM e as variantes genéticas dos componentes do inflamassoma têm sido associadas a doenças autoimunes e neurodegenerativas, com isso hipotetizamos que o inflamassoma e suas citocinas IL-1Beta e IL-18, podem representar importantes contribuintes na patogênese da EM e eventualmente explicar, pelo menos em parte, a heterogeneidade observada em pacientes com EM. Fizemos uma análise multivariada que foi realizada com base na forma clínica (recorrente remitente/RR, primária progressivo /PP ou secundário progressiva /SP, índice de gravidade (EDSS) e índice de progressão (IP). Os monócitos do sangue periférico (PBMC) dos pacientes foram examinados para ativação do inflamassoma (Produção de IL-1Beta e IL-18, clivagem de caspase-1). Com os objetivos de avaliar a contribuição do inflamassoma na EM, em termos de (a) efeito genético sobre o desenvolvimento, gravidade e / ou prognóstico, e (b) ativação complexa de células de sangue periférico como uma forma de avaliar a inflamação sistêmica. Para isso, utilizamos variantes genéticas funcionais em componentes do inflamassoma, que foram analisadas em uma coorte de pacientes com EM, pelo uso de ensaios específicos de alelos e qPCR. A analise multivariada resultou em associação com a variante -511C / T IL1B ganho de função, sendo essa mais frequente em formas progressivas (especialmente SP) do que em RR. A variante de ganho de função NLRP3 Q705K resultou mais frequente em pacientes com EDSS > 3 do que em pacientes com EDSS < 3 e, consequentemente, esse SNP está associado a um IP mais elevado. A análise de PBMC mostrou que as células de indivíduos EM, são mais propensas a responder a um estímulo NLRP3 clássico (isto é, LPS) do que as dos doadores saudáveis. Em conjunto, esses achados indicaram que os pacientes com EM apresentam uma desregulação no inflamassoma NLRP3, podendo ser avaliada no sangue periférico facilitando um prognóstico, e que esse perfil pode ser secundário a um mecanismo genético pró-inflamassoma / The multiple sclerosis (MS), neurodegenerative disease of the central nervous system (CNS) with autoimmune and inflammatory characteristics, with initial events, as well as the evolution of MS, are heterogeneous (three main clinical forms) and multifactorial. Innate immunity has recently been shown to be an important factor in MS and the genetic variants of the components of inflammassoma have been associated with autoimmune and neurodegenerative diseases, thereby hypothesizing that the inflammassoma and its IL-1Beta and IL-18 cytokines may represent important contributors in the pathogenesis of MS and possibly explain, at least in part, the heterogeneity observed in MS patients. We performed a multivariate analysis that was performed based on clinical form (recurrent recurrent / RR, progressive primary / PP or progressive secondary / SP, EDSS and progression index.) Peripheral blood mononuclear cells (PBMC) of patients were examined for inflammatory activation (IL-1Beta and IL-18 production, caspase-1 cleavage). With the objectives of evaluating the contribution of inflammassoma in MS in terms of (a) genetic effect on development, severity and / or prognosis, and (b) complex activation of peripheral blood cells as a way of assessing systemic inflammation. For this, we used functional genetic variants in components of the inflammassoma, which were analyzed in a cohort of MS patients, through the use of specific allele and qPCR assays. For this, we used functional genetic variants in components of the inflammassoma, which were analyzed in a cohort of MS patients, through the use of specific allele and qPCR assays. Multivariate analysis resulted in association with the -511C / T IL1B function gain, which is more frequent in progressive forms (especially SP) than in RR. The gain variant of NLRP3 Q705K function was more frequent in patients with EDSS > 3 than in patients with EDSS < 3 and, consequently, this SNP is associated with a higher PI. PBMC analysis showed that cells from MS individuals are more likely to respond to a classical NLRP3 (ie LPS) stimulus than healthy donors. Taken together, these findings indicated that patients with MS have a dysregulation in the NLRP3 inflammassoma and can be evaluated in the peripheral blood facilitating a prognosis and that this profile may be secondary to a pro-inflammatory genetic mechanism

Le développement d’un cancer chez un individu immunocompétent témoigne del’inefficacité de ses défenses immunitaires naturelles à entraver la progression tumorale. Larestauration ou l’induction de réponses immunitaires anti-tumorales efficaces sont des enjeuxmajeurs des stratégies thérapeutiques actuelles. En plus des immunothérapies classiques,visant à activer le système immunitaire des patients contre leurs tumeurs, les thérapiesconventionnelles, telles que les chimiothérapies et la radiothérapie, peuvent avoir des effetsimmunostimulants, en parallèle de leur effet directement cytotoxique sur les cellulestumorales. Parmi celles-ci, les anthracyclines, l’oxaliplatine et la radiothérapie sontnotamment capables d’induire une mort cellulaire immunogène. L’efficacité thérapeutique deces traitements dépend de l’activation de lymphocytes T (LT) CD8 anti-tumoraux,producteurs d’interféron-gamma (IFN-γ). En utilisant des modèles murins de tumeurstransplantées, nous montrons que le traitement des cellules tumorales par des droguesimmunogènes induit la libération d’adénosine triphosphate (ATP) dans le milieu extracellulaire.Ce signal de danger endogène se lie à son récepteur P2RX7 sur les cellulesdendritiques et permet la formation de l’inflammasome NLRP3/ASC/pro-caspase 1,conduisant à l’activation protéolytique de la caspase 1, qui active la pro-interleukine-1β (pro-IL-1β) en interleukine-1β (IL-1β). L’IL-1β peut directement agir sur les LT CD8 pourfaciliter leur polarisation vers un phénotype de lymphocytes T cytotoxiques de type 1 (Tc1).De plus, le traitement de tumeurs établies par des drogues immunogènes induit uneproduction d’IL-17A (IL-17) dans le lit tumoral, au cours de la première semaine suivant letraitement. Les LT gamma delta (LTγδ) sont les principaux producteurs de cette cytokinedans nos modèles. Ces cellules s’accumulent transitoirement dans la tumeur dès le troisièmejour suivant le traitement et leur taux est parfaitement corrélé à celui des LT CD8 producteursd’IFN-γ tumoraux. Comme l’IFN-γ, l’IL-17 et les LTγδ sont nécessaires à l’efficacitéthérapeutique des drogues immunogènes. L’IL-1β produite par les cellules dendritiquesexposées à des corps apoptotiques immunogènes stimule directement la production d’IL-17par les LTγδ. Ces travaux ont contribué à identifier l’ATP extra-cellulaire comme undéterminant moléculaire de l’immunogénicité des cellules tumorales exposées à certainesthérapies anti-cancéreuses conventionnelles. De plus, nous montrons que les traitementscytotoxiques classiques peuvent moduler l’environnement immunitaire dans le lit tumoral, etcontribuer à remplacer une inflammation chronique et immunosuppressive en uneinflammation aigüe et immunogène. L’étude des interactions entre le système immunitaire etles thérapies anti-cancéreuses conventionnelles est nécessaire pour utiliser ces traitements demanière plus rationnelle, en les combinant par exemple à des immunothérapies plusclassiques. / In most cases, cancer occurs in immunocompetent individuals, revealing the potential failure of the immune system to impair tumor development. Trying to restore or to induce an efficient anti-tumoral immune response is part of the main challenges of cancer treatment. In addition to the classical immunotherapeutic compounds, conventionnal cytotoxic treatments can also have some immunostimulatory effects. Among these treatments, the chemotherapeutic agents oxaliplatin and anthracyclins, as well as irradiation, can induce an immunogenic cell death, contributing to the activation of IFNγ-producing CD8 T cells. We show that upon treatment with immunogenic drugs, tumor cells are able to release ATP in the extracellular medium. This ATP can act on dendritic cells through its receptor P2RX7, which is able to activate the inflammasome NLRP3 and the subsequent production of IL-1β. Using models of transplantable tumors in mice we could show that the production of IL-1β induced by immunogenic treatments was mandatory for their efficacy. In addition to being directly involved in Tc1 cells polarization, IL-1β could promote the production of IL-17A by gamma delta T cells which were accumulating in the tumor bed following chemotherapy. We could show that these IL-17 producing gamma delta T cells were involved in treatment efficacy. This work has revealed that extracellular ATP was a crucial endogenous danger signal involved in anti-cancer chemotherapy efficacy. Furthermore, we have shown that conventionnal cytotoxic compounds could transform the smoldering pro-tumoral inflammation into an acute and immunogenic anti-tumoral one. This work may have some clinical implications, especially in the setting of therapeutic protocols involving the combination of conventionnal cytotoxic therapies with selected immunoadjuvants for the treatment of cancer.

L’inflammation joue un rôle clé dans de nombreuses pathologies, telles que les maladies inflammatoires chroniques, les désordres métaboliques et le cancer. L’un de ses médiateurs le plus puissant est l’interleukine-1β (IL-1β), qui est une cytokine pro-inflammatoire participant à tous les stades de l’inflammation et de l’immunité. Son activation est régulée par un complexe multi-protéique nommé inflammasome, dont la caspase-1 active en découlant est responsable du clivage et de la maturation de l’IL-1β. Huit types d’inflammasomes activant et clivant la pro-caspase-1 ont été identifiés et contiennent tous la protéine ASC (apoptosis-associated speck-like protein containing a CARD). Les inflammasomes partagent un signal intracellulaire commun et le mécanisme menant à leur assemblage et leur activation n’est pas totalement élucidé. L’utilisation de la lignée cellulaire humaine monocytaire, THP-1, différenciée en macrophages grâce à un ester de phorbol, le TPA, a permis la mise en place d’un modèle d’étude de modulateurs de l’inflammasome en conditions stériles. Ce modèle a permis l’étude des mécanismes impliqués suite à des signaux issus de l’inflammation chronique, tels que l’ATP et les espèces réactives de l’oxygène (ROS). Ce travail montre qu’il existe une synergie entre ATP et ROS, qui agissent grâce à une boucle d’activation impliquant probablement plusieurs inflammasomes, dont NLRP3. Des donneurs de NO connus (trinitrine et isosorbide dinitrate) ou nouveau (dérivé de purine) ont montré une activité anti-inflammatoire. D’autres composés ont été identifiés comme de potentiels inhibiteurs d’inflammasome (extraits de dattes et dérivé de purine portant un acide lipoïque) / Inflammation has a pivotal role in several disorders, such as chronic inflammatory diseases, metabolic disorders and cancer. Interleukin-1β (IL-1β) is one of the most powerful mediators in this mechanism. IL-1β is a pro-inflammatory cytokine which is implicated in every step of inflammation and immunity. IL-1β is regulated by a multi-protein complex named inflammasome, in which active caspase-1 is responsible for IL-1β processing and maturation. Eight inflammasomes processing and activating pro-caspase-1 has been identified and all contain the adaptor protein ASC (apoptosis-associated speck-like protein containing a CARD). Inflammasomes share a common intracellular signal and the mechanism for their structural assembly and activation is not totally clear. The human monocytic cell line, THP-1, has been used and cells have been differentiated in macrophages by the phorbol ester, TPA. Based on this, a cellular pattern has been established for studying inflammasome modulators under sterile conditions. This pattern allowed the study of implicated mechanisms in chronic inflammatory signals, such as ATP and reactive oxygen species (ROS). This study shows that ATP and ROS synergize for activating inflammasomes and NLRP3 and act in an autocrine loop. Stimulation with others natural or synthetic compounds has been realized for characterization of their inflammatory activity. Therapeutic NO donors (trinitrin and isosorbide dinitrate) and a new NO donor (purine nitrate ester) have been characterized for their anti-inflammatory activity. Others compounds from a new category of inflammasome inhibitors have been identified (date fruit extracts and lipoic acid purine derivative)