Inflammation in the absence of infection (sterile inflammation) is a crucial host defence response to tissue injury, but is also considered to contribute to the pathogenesis of many diverse disease states, including stroke. Sterile inflammation is initiated by damage associated molecular patterns (DAMPs) which are endogenous molecules released from necrotic cells or that are modified during disease. The pro-inflammatory cytokines IL-1α and IL-1β are key mediators of inflammation. IL-1β release is controlled by caspase-1 which, in turn, is regulated by the inflammasome. The NOD-, LRR-, pyrin domain-containing 3 (NLRP3) inflammasome is most typically associated with sterile inflammation and the recognition of DAMPs. Thus, understanding the mechanisms of NLRP3-activating DAMP-induced inflammation may lead to the identification of novel therapeutic targets with which to treat inflammatory diseases. This thesis sought to determine how NLRP3-activating DAMPs affect the pro-inflammatory response of glia, the immune cells of the brain. Experimental models in vitro typically use a pathogen associated molecular pattern (PAMP) such as LPS to prime cells before observing their response to NLRP3-activating DAMPs. As the brain is protected by the blood brain barrier (BBB), it is unlikely glia would be exposed to PAMP priming. However it remains unclear as to how glia respond to NLRP3-activating DAMPs in the absence of priming, or what the source of endogenous priming is. Therefore, the initial hypothesis was to investigate the pro-inflammatory response of mixed glia in vitro to NLRP3-activating DAMPs in the absence of PAMP priming. It is shown here for the first time that NLRP3-activating DAMPs can initiate an IL-1-NLRP3-independent inflammatory response in mixed glia in the absence of PAMP priming. Moreover, it is shown that the acute phase protein serum amyloid A is elevated in plasma after stroke and may act as an endogenous priming signal to allow IL-1β-dependent inflammation to contribute to the damage after breakdown of the BBB.Inflammation following acute sterile injury such as stroke is augmented by persisting cell death. It was therefore hypothesised that NLRP3-activating DAMPs released after the initial injury, may initiate a form of programmed cell death that continues to drive inflammation. Using inhibitors of specific types of cell death, it was identified that NLRP3-activating DAMP induced cell death is likely to be necrosis and not programmed cell death. Further investigation into the biological importance of DAMP-induced IL-1-independent inflammation and the specific contribution of acute phase proteins to brain pathology may aid the identification of new therapeutic targets.

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While cognitive impairment is common in peripheral diseases such as chronic kidney disease (CKD), mechanistic insights and effective therapies are lacking. Multiple toxins accumulating as a consequence of CKD have been identified, yet the consequences for cellular crosstalk in the brain and the mechanisms underlying the associated neuronal dysfunction remain largely elusive. In the case of CKD, more than 100 uremic toxins have been identified. Renal transplantation largely reverses the cognitive impairment associated with CKD, demonstrating that cognitive impairment in CKD can be reversed. This indicates that pharmaceutical approaches to target cognitive impairment in association with CKD may be feasible. However, it is unlikely that targeting a single toxin will be sufficient to combat neuronal dysfunction associated with peripheral diseases such as CKD, given the large number of toxins involved and since the pattern of accumulating toxins varies among affected patients. Rather than identifying single toxins, identifying a common mechanism inducing neuronal dysfunction and thus impairing cognition may identify new and feasible therapeutic approaches. One commonality of peripheral diseases such as liver or renal failure is sterile inflammation. Sterile inflammation has been linked with neurodegenerative diseases and associated cognitive impairment and inflammasome activation is one hallmark of chronic pathologies in the brain. Mutations in the inflammasome component NLRP3 show clinical manifestations of cryopyrin- associated periodic syndromes (CAPS), which are characterized by skin rash, fever and joint pain. Further, abnormal and constant NLRP3 signaling has been associated with some chronic and degenerative diseases such as Alzheimer’s disease (AD), atherosclerosis, arthritis or cancer. A causative function of the NLRP3 inflammasome for neurodegenerative processes is supported by preclinical studies. These pre-clinical studies used whole body knock out mice to demonstrate that deficiencies of NLRP3, caspase-1 or the primary receptor for IL-1β, IL-1R1, protect mice from neurodegenerative processes. While providing important insights into the role of the NLRP3-inflammasome in neurodegenerative processes, these studies did not identify the relevant cell types in which the inflammasome is activated, the mechanisms underlying inflammasome activation and the consequences thereof, e.g. for intracerebral cross-talk. In addition, whether sterile inflammation triggered by the NLRP3 inflammasome impairs cognition in the setting of primarily peripheral diseases such as CKD remains unknown. To address these open questions, I used a mouse model of CKD, in which I detected NLRP3 inflammasome in brains. Interestingly, despite inflammasome activation in the brain, microglial caspase-1 deficiency did not improve cerebral inflammation and cognition in CKD mice. I identified noncanonical IL-1β maturation in microglia in CKD conditions, which was cathepsin c – caspase-8 mediated. Restoring K+ homeostasis in microglia or genetic inhibition of neuronal IL-1R1 signaling abolished CKD-induced cognitive impairment. Mechanistically, noncanonical IL-1β maturation and secretion from microglia promotes via IL-1R signaling cognitive impairment in neurons. This identifies a molecular mechanism of sterile CNS inflammation and the associated intercellular signaling pathway, which may be therapeutically amendable. Microglial K+ dyshomeostasis and noncanonical microglial IL-1β maturation may be druggable targets in some forms of cognitive impairment.:Content 2 List of abbreviations 5 Graphical abstract 8 2 Introduction 9 2.1 Chronic kidney disease and cognition 11 2.2 Microglia cells 13 2.3 The inflammasome, potassium dyshomeostasis in brain cells and thallium autometallography 15 2.4 Sterile inflammation in neurodegenerative diseases 17 3 Aims of the study 19 4 Materials and Methods 20 4.1 Reagents 20 4.2 Mice 27 4.3 CKD mouse model (5/6 nephrectomy model) 30 4.4 Evans Blue extravasation assay 32 4.5 2-photon microscopy 32 4.6 Analysis of mice 33 4.7 In vivo interventions 33 4.8 Histology and immunohistochemical analysis 34 4.9 Cell culture 34 4.10 Dextran permeability assay 35 4.11 Thallium-AMG (TlAMG), ex vivo and in vitro 36 4.12 Protein extraction and Western blotting 38 4.13 IL-1β ELISA 38 4.14 Reverse Transcriptase Polymerase Chain Reaction (RT–PCR) 38 4.15 Proximity ligation assay (PLA) 39 4.16 Behavioral analysis 39 4.17 Cathepsin c substrate assay 40 4.18 snRNA-Seq 41 4.19 Statistical Analysis 42 5 Results 43 5.1 Chronically impaired renal function leads to cognitive decline 43 5.2 Blood brain barrier (BBB) disruption in chronic kidney disease 44 5.3 Potassium dyshomeostasis in brain cells in CKD 45 5.4 CKD leads to microglia activation 46 5.5 Priming of microglia in CKD depends on potassium dyshomeostasis and its restoration improves cognition in CKD 49 5.6 TRAM34 ameliorates potassium dyshomeostasis and behavior in CKD 51 5.7 Uremia-induced cognitive impairment depends on microglia- neuron crosstalk via IL-1R1 52 5.8 Deciphering the microglial molecular pathway in CKD 56 5.9 Microglia activation in CKD is independent of NLRP3 56 5.10 Microglial IL-1β maturation occurs independently of the NLRP3-Caspase-1 inflammasome in CKD 57 5.11 The role of caspase- 8 in microglia activation in CKD 60 5.12 Lysosomal cathepsin c promotes microglia activation pivotal for caspase-8 activation 62 5.13 Broader implication of the pathway in other chronic peripheric diseases 63 5.14 Microglia inflammasome activation and IL-1β release is sufficient to induce cognitive impairment 64 5.15 Tables 66 6 Discussion 69 7 Summary 75 8 Zusammenfassung 80 9 References 86 10 Declaration about the independent preparation of the work 97 11 Presentation of own contribution 98 12 Curriculum vitae 99 13 Publications 104 14 Acknowledgments 106

Les LT CD8 mémoires constituent une population hétérogène. Une modulation des différents signaux d’activation des LT CD8 naïfs influe sur la diversité des LT CD8 effecteurs et mémoires générés. A coté des sous populations classiquement décrites de LT CD8 mémoires développés dans des conditions infectieuses, il existe une population de LT CD8 mémoires générés dans des conditions d’inflammation stérile, c’est-à-dire en absence de pathogènes et de signaux moléculaires dérivés de pathogènes : les TIM pour « T-inflammatory memory cells ». Au cours de cette thèse j’ai eu pour objectif : 1) de mieux caractériser la population de TIM 2) d’évaluer leur capacité à répondre à une activation par un pathogène viral et ainsi leur plasticité et 3) d’étudier leur recrutement au site d’une infection locale respiratoire.J’ai ainsi pu identifier de nouvelles propriétés des TIM et montrer que les TIM sont recrutés dans une réponse secondaire à un virus exprimant la même spécificité antigénique, en générant des LT CD8 mémoires secondaires aux fonctions améliorées. De plus, les LT CD8 mémoires secondaires générés présentent un avantage fonctionnel par rapport aux LT CD8 mémoires primaires dans leur capacité de production de TNF-α et de la chimiokine XCL1. Cette dernière propriété pourrait leur conférer un avantage pour la réponse à des antigènes cross présentés. J’ai par ailleurs montré la capacité des TIM à être recrutés au niveau du poumon et des voies aériennes au cours d’une infection respiratoire virale. Ces résultats montrent que les LT CD8 mémoires générés dans des conditions d’activation inflammatoires stériles peuvent prendre part au contingent de cellules immunitaires impliquées dans des réponses à des infections. Ces résultats ouvrent un champ d’investigation intéressant concernant la plasticité des LT CD8 mémoires. Ils pourraient avoir une incidence sur certaines pathologies inflammatoires, dans le cas d’une ré-activation des TIM par un virus cross réactif. / Memory CD8 T cells represent a heterogeneous population. A modulation of the activation signals during naïve CD8 T cells activation influences the diversity of the Effector and Memory CD8 T cells generated. Besides the classically described subsets of Memory CD8 T cells, generated under infectious conditions, are T inflammatory memory CD8 T cells (TIM). TIM are generated under sterile priming conditions that are devoid of pathogens and pathogen-derived danger signals.During this thesis, I had the latter objectives: 1) a better characterisation of TIM, 2) to evaluate whether they could be recruited in an immune response directed against a virus and thus investigate their plasticity, 3) to examine their recruitment to the site of a respiratory local infection.I have identified new features of TIM and shown that TIM can take part to the immune response triggered by a virus expressing their cognate antigen and further differentiate into secondary memory cells with improved functions. The secondary memory CD8 T cells they generate display a functional advantage over primary memory cells in their capacity to produce TNF-α and the XCL1 chemokine. This last result could give them an advantage against cross-presented antigen. Furthermore, I have shown that TIM cells can be recruited in lung and airways during a respiratory viral infection. These results show that memory CD8 T cells generated under sterile priming conditions can take part to the contingent of immune cells involved in responses to infection. They open an interesting field of investigation of the plasticity of memory CD8 T cells. They could have an impact in inflammatory diseases, in the case of re-activation of TIM by a cross-reactive virus.

A diabetes tipo 1 (DT1) está associada `a inflamação estéril (IE) e maior susceptibilidade a sepse. A sepse induz a síndrome da resposta inflamatória sistêmica (SIRS) e a inflamação pulmonar aguda (ALI). O leucotrieno (LT) B4 produzido condições inflamatórias induz a expressão de MyD88 em macrófagos (MA). Hipotetizamos que a DT1 induz a síntese de LTB4 promovendo a IE e isto contribui para SIRS, susceptibilidade a sepse e ALI. Os diabéticos apresentaram níveis elevados de LTB4 e IL-1b no soro e seu MA expressaram mais MyD88/STAT-1. A expressão de STAT-1 foi induzida por c-Jun de forma dependente de LTB4. O tratamento com insulina restaurou os níveis de LTB4 e STAT-1/MyD88 e a inibição de LTB4 restaurou os níveis de MyD88 e IL-1b. Na sepse, a inibição de 5LO prolongou a sobrevida dos diabéticos e diminuiu a SIRS menos IL-1b e IL-10 no soro e TNF-a e IL-1b na cavidade peritoneal. O pulmão dos diabéticos apresentaram ALI menos intensa que se correlacionou com um altos níveis de SOCS-1, baixos níveis de MyD88 e falha na ativação de NFkB nos macrófagos alveolares. / Type 1 diabetes (T1D) is associated with sterile inflammation (SI) and increased sepsis susceptibility. Sepsis induces Systemic Inflammatory Response Syndrome (SIRS) and Acute Lung Injury (ALI). Leukotriene (LT) B4 is produced in inflammatory conditions and induces MyD88 expression in macrophages (MA). We hypothesized that T1D induce LB4 that promotes SI contributing to SIRS, sepsis susceptibility and ALI. Diabetics presented higher levels of LTB4 and e IL-1b in the serum and MA expressed more MyD88/STAT-1. STAT-1 expression was induced by c-Jun on LTB4 dependent manner. Insulin treatment restored LTB4 and STAT-1/MyD88 levels and inhibition of LTB4 restored MyD88 and IL-1b levels. During sepsis, 5LO inhibition increased diabetics survival and inhibited SIRS- lower levels of IL-1b and IL-10 in the serum and TNF-a and IL-1b in the peritoneal cavity. Lungs from diabetics presented milder ALI that correlated with high levels of SOCS-1, low levels of MyD88 and impaired NFkB activation in alveolar macrophages.

A diabetes tipo 1 (DT1) está associada `a inflamação estéril (IE) e maior susceptibilidade a sepse. A sepse induz a síndrome da resposta inflamatória sistêmica (SIRS) e a inflamação pulmonar aguda (ALI). O leucotrieno (LT) B4 produzido condições inflamatórias induz a expressão de MyD88 em macrófagos (MA). Hipotetizamos que a DT1 induz a síntese de LTB4 promovendo a IE e isto contribui para SIRS, susceptibilidade a sepse e ALI. Os diabéticos apresentaram níveis elevados de LTB4 e IL-1b no soro e seu MA expressaram mais MyD88/STAT-1. A expressão de STAT-1 foi induzida por c-Jun de forma dependente de LTB4. O tratamento com insulina restaurou os níveis de LTB4 e STAT-1/MyD88 e a inibição de LTB4 restaurou os níveis de MyD88 e IL-1b. Na sepse, a inibição de 5LO prolongou a sobrevida dos diabéticos e diminuiu a SIRS menos IL-1b e IL-10 no soro e TNF-a e IL-1b na cavidade peritoneal. O pulmão dos diabéticos apresentaram ALI menos intensa que se correlacionou com um altos níveis de SOCS-1, baixos níveis de MyD88 e falha na ativação de NFkB nos macrófagos alveolares. / Type 1 diabetes (T1D) is associated with sterile inflammation (SI) and increased sepsis susceptibility. Sepsis induces Systemic Inflammatory Response Syndrome (SIRS) and Acute Lung Injury (ALI). Leukotriene (LT) B4 is produced in inflammatory conditions and induces MyD88 expression in macrophages (MA). We hypothesized that T1D induce LB4 that promotes SI contributing to SIRS, sepsis susceptibility and ALI. Diabetics presented higher levels of LTB4 and e IL-1b in the serum and MA expressed more MyD88/STAT-1. STAT-1 expression was induced by c-Jun on LTB4 dependent manner. Insulin treatment restored LTB4 and STAT-1/MyD88 levels and inhibition of LTB4 restored MyD88 and IL-1b levels. During sepsis, 5LO inhibition increased diabetics survival and inhibited SIRS- lower levels of IL-1b and IL-10 in the serum and TNF-a and IL-1b in the peritoneal cavity. Lungs from diabetics presented milder ALI that correlated with high levels of SOCS-1, low levels of MyD88 and impaired NFkB activation in alveolar macrophages.

No description available.

Die intrazelluläre Weiterleitung von Interferonsignalen von der Zytoplasmamembran zum Zellkern wird vermittelt über den Signaltransduktor und Aktivator der Transkription 1 (STAT1), welcher in seiner tetrameren Form als Transkriptionsfaktor an Immunantworten beteiligt ist. In diesem Projekt wurde der Protomerenaustausch zwischen STAT1-Dimeren unter kinetischen Gesichtspunkten untersucht und dabei dieser Prozess als ein potentiell geschwindigkeitsbestimmender Schritt des Aktivierungs-/Inaktivierungs-Zyklus von STAT1 identifiziert. Die Daten unterstützen einen alternativen Mechanismus für den Wechsel zwischen der parallelen und antiparallelen Konformation von STAT1-Dimeren basierend auf der Dissoziation und nachfolgenden Reassoziation von Protomeren, bei dem reziproke Interaktionen innerhalb des N-terminalen Domänendimers zur Stabilisierung eines intermediären Konformationsübergangs nicht benötigt werden. Durch Bindung an spezifische DNA-Zielbereiche, als Gamma-aktivierte Sequenzen (GAS) bezeichnet, wird die Dynamik des Protomerenaustauschs wesentlich beeinträchtigt. In der Sequenz des für das zytoskelettale Strukturprotein Ezrin kodierenden humanen EZR-Gens wurde mittels in silico Analyse ein doppeltes GAS-Motiv als mögliche STAT1-Zielsequenz identifiziert und die Bindung von STAT1-Dimeren an jedes der beiden Elemente sowie eine moderate Geninduktion bestätigt. Allerdings zeigen Mäuse mit einer N-terminalen Substitutionsmutation von STAT1, welche die kooperative DNA-Bindung beeinträchtigt, sowie auch ein kompletter funktioneller Knockout des Stat1-Gens keine veränderte Expression von Ezrin und Moesin in Knochenmarkszellen verglichen mit Mäusen, die das Wildtyp-Molekül exprimieren. In einem Myokardinfarktmodell durch Ligatur des Ramus interventricularis anterior zeigen männliche Mäuse mit Expression der Interferon-γ-irresponsiven STAT1-Mutante höhere Überlebensraten, während weibliche Tiere vor den nachteiligen Effekten des kardialen Remodellings in der frühen Phase geschützt sind. In entzündlichen myokardialen Infiltraten dieser Tiere wurde ein geringfügig höheres Expressionsniveau an tyrosinphosphoryliertem STAT1 nachgewiesen, während die Gesamtproteinmenge an STAT1 gegenüber dem Wildtyp reduziert war. Zellen aus lymphatischen Organen STAT1-defizienter Tiere mit experimenteller autoimmuner Enzephalomyelitis, die als Modell einer T-Helfer-Zell-vermittelten Autoimmunerkrankung verwendet wurde, zeigten einen hyperproliferativen Phänotyp und sezernierten größere Mengen an IFNγ und IL-17A. Injektion dieser Mäuse mit Lipopolysaccharid während der Induktionsphase der experimentellen autoimmunen Enzephalomyelitis hob den hyperproliferativen Phänotyp vollständig auf. Zusammenfassend demonstrieren die Ergebnisse aus dieser Arbeit die Bedeutung einer kooperativen DNA-Bindung und Tetramerstabilisierung von STAT1 im Zusammenspiel komplexer immunologischer Prozesse auch in Abwesenheit infektiöser Pathogene und unterstreichen zudem die Schlüsselrolle von tyrosinphosphoryliertem STAT1 bei der Verknüpfung zwischen angeborenem und erworbenem Immunsystem.

La voie des RLR (RIG-I like Receptors) joue un rôle essentiel dans la détection des virus à ARN, déclenchant une réponse immunitaire antivirale grâce au recrutement de la protéine adaptatrice mitochondriale MAVS (Mitochondrial AntiViral Signaling protein). Nous avons mis en évidence que les souris déficientes pour la protéine MAVS (MAVS KO) présentaient un phénotype proallergénique dans un modèle d'inflammation stérile d'hypersensibilité retardée de contact (HSRC) qui reproduit la dermatite allergique de contact (DAC) chez l'homme. Nous avons caractérisé le système immunitaire des souris MAVS KO en condition d'équilibre et durant la réponse d'HSRC. Nous avons identifié un rôle du microbiote intestinal des souris MAVS KO dans l'exacerbation de réponse d'HSRC et mis en évidence une dysbiose du microbiote bactérien. Nous avons démontré que la dysbiose était responsable du phénotype inflammatoire observé, phénotype transmissible à des souris sauvages par des expériences de cohébergement et de transplantation fécale. Cette dysbiose induit une augmentation de la perméabilité intestinale chez les souris MAVS KO lors de la réponse d'HSRC, aboutissant à une translocation bactérienne dans les organes lymphoïdes et à la modulation des réponses immunitaires à l'origine de l'exacerbation de réponse d'hypersensibilité. La 2ème partie de ma thèse vise à étudier les conséquences de la déficience en MAVS sur le métabolisme glucidique. Nos expériences ont démontré que les souris MAVS KO développaient une surcharge pondérale et une insulino-résistance sous régime riche en lipides et sucrose, dépendants de la dysbiose intestinale. Au niveau cellulaire, une altération des interactions aux points de contact entre la mitochondrie et le réticulum endoplasmique a été observée. Nos résultats permettent d'envisager le développement de nouvelles approches thérapeutiques des pathologies allergiques et métaboliques humaines par la modulation du microbiote intestinal / RIG-I like receptors (RLRs) play a major role in response to cytosolic viral RNAs by initiating an antiviral immune response through the recruitment of the mitochondrial adaptor protein MAVS (Mitochondrial AntiViral Signaling protein). We showed that MAVS-deficient mice developed an exacerbated response in a sterile inflammatory model of Contact Hypersensitivity (CHS), that reproduces the pathophysiology of allergic contact dermatitis (ACD) in human. We characterized the immune system of MAVS KO mice at steady state and during CHS response. We found that MAVS deficiency leads to changes in the gut bacterial composition suggesting an unexpected role of the RLR pathway in the regulation of intestinal homeostasis. We demonstrated that intestinal dysbiosis is responsible for the increased CHS response, and showed that the inflammatory phenotype of MAVS KO mice can be transferred to WT mice by cohousing and fecal transplantation. We demonstrated that the dysbiotic gut microbiota exerts its effect due to an increased intestinal permeability during DTH sensitization. The ensuing bacterial translocation within lymphoid organs enhances characteristic cytokines production that increases CHS response. The 2nd part of my thesis aimed to study the consequences of MAVS deficiency on glucose metabolism. Our experiments showed that MAVS KO mice exhibit disorders of glucose homeostasis during high fat diet (HFD) associated with the development of overweight and insulin resistance. We also observed alterations of MAM (Mitochondria-associated endoplasmic reticulum membranes), contact poins between mitochondria and endoplasmic reticulum. Recent preliminary data suggested that the metabolic disorders associated with MAVS deficiency are due to intestinal dysbiosis. Our results highlight a new role for the RLR pathway and allow to consider the development of new therapeutic approaches to human allergic and metabolic diseases by modulation of the intestinal microbiota