Coordenação de Aperfeiçoamento de Pessoal de Nível Superior / Glutaric acidemia type I (GA-I) is an inborn error of metabolism (EIM), characterized biochemically by major accumulation of glutaric acid (GA) and pathologically by a characteristic striatal degeneration. The clinical manifestations are mainly neurological and develop during childhood (up to 5 years old). Among these changes, there are the seizures and cognitive deficits, which may be precipitated by infectious processes. From this, the first hypothesis to be tested in this study was to investigate whether lipopolysaccharide E. coli 055 B5 serotype (LPS; 2 mg/Kg; i.p.), an inflammatory agent, could facilitate seizures induced by GA in young rats (21 days of life). For this, firstly it was determined the acute dose of intrastriatal GA (1.3 μmol/striatum) that cause behavioral and electroencephalographic (EEG) seizures in young rats. Moreover, it was shown that LPS administration 3 hours before GA intrastriatal injection did not change the seizures, but when LPS was administered 6 hours before the GA, it reduced the latency and increased the duration of behavioral and EEG seizures induced by GA in young rats. It also was observed that LPS injection caused an initial drop in rectal temperature of young rats (up to 2 hours), followed by a rise in temperature that started at 3 hours and remained high until 6 hours after LPS injection. Furthermore, it was shown that LPS injection 3 and 6 hours before intrastriatal injection of GA caused an increase in striatal levels of IL-1β in young rats, and this increase was statistically higher in 6 than in 3 hours. In addition, it was observed that the increase in IL-1β striatal levels, caused by LPS administration, positively correlated with total time of seizures. Finally, it was observed that previous use of IL-1β antibody prevented the latency reduction and the increased duration of seizures caused by LPS administration 6 h before intrastriatal injection of GA in young rats. Thus, these findings suggest that the signaling of IL-1β present in inflammation produced by LPS contributes significantly to neuronal hyperexcitability, and thus to reduce latency and increase the duration of seizures induced by GA. Therefore, pharmacological treatments that block the specific functions or overproduction of IL-1β in GA-I, may represent an unconventional strategy to treat this condition. However, clinical studies should be conducted to evaluate the effectiveness of treatment in glutaricoacidemic patients with convulsions. Since patients with GA-I have other important neurological changes addition to the seizures, as cognitive impairments, the second hypothesis to be tested in this study was to determine whether chronic treatment with GA (5 μmol/g; s.c.; twice per day; from the 5th to the 28th day of life) could cause spatial memory impairment in young rats, and verify whether the inflammation produced by LPS (2 mg/Kg; i.p.; one per day; from the 25th to the 28th day of life) could facilitate the cognitive deficit induced by GA. In addition, it also was evaluated the possible impact of these treatments on functional and structural changes in the hippocampus of these animals. Initially it was shown that chronic treatment with GA, as well as the treatments with LPS and GA-LPS, caused a deficit in spatial learning of young rats. However, it was demonstrated that the treatment with GA-LPS produced a greater impairment in spatial memory compared to other treatments. In addition, it was observed that none of the treatments affected weight or locomotor activity/exploratory of animals. It also was shown that chronic treatment with GA, as well as treatments with LPS and GA-LPS, increased the hippocampal levels of IL-1β and TNF-α in young rats. Furthermore, it was demonstrated that treatments with GA, LPS and GA-LPS caused a reduction in total hippocampal volume of young rats. Finally it was observed that treatments with GA, LPS and GA-LPS caused a reduction of α1 subunit activity of Na+,K+-ATPase enzyme. On the other hand, it was shown that treatments with GA and LPS caused an increase in activity of α2/3 subunits of the enzyme. Thus, only treatment with GA-LPS showed a reduction in total activity of Na+,K+-ATPase in the hippocampus of young rats. These data indicate that the impairment in spatial learning observed in rats treated with GA, LPS and GA-LPS was due to increased levels of inflammatory cytokines, the reduction in hippocampal volume and the inhibition of α1 subunit activity of Na+,K+-ATPase enzyme. However, the worsening in spatial memory observed in rats treated with GA-LPS was due to inhibition of total activity of Na+,K+-ATPase, which was specific α2/3 isoforms, since only this group showed no compensatory response the activity of these subunits. Therefore, this second part of the study showed that chronic treatment with GA caused a deficit in spatial learning in young rats, and that the presence of an inflammatory process increased the impairment in spatial memory induced by GA alone. Thus, understanding the mechanisms involved in seizures and cognitive deficits observed in patients with GA-I in the presence of an inflammatory process is important for the development of new therapies to treat this condition, as well as other diseases associated with the presence of inflammatory mediators. / A acidemia glutárica tipo I (GA-I) é um erro inato do metabolismo (EIM) caracterizada bioquimicamente pelo acúmulo principal de ácido glutárico (GA) e patologicamente por uma característica degeneração estriatal. As manifestações clínicas são predominantemente neurológicas, e desenvolvem-se principalmente na infância (até os 5 anos de idade). Entre estas alterações, destacam-se as convulsões e os déficits cognitivos, os quais podem ser precipitados por processos infecciosos. A partir disso, a primeira hipótese a ser testada neste estudo foi investigar se o lipopolissacarídeo sorotipo E. coli 055 B5 (LPS; 2 mg/Kg; i.p.), um agente inflamatório, facilitaria as convulsões induzidas pelo GA em ratos jovens. Para isso, primeiramente determinou-se a dose intraestriatal aguda de GA (1.3 μmol/estriado) que causa convulsões comportamentais e eletroencefalográficas (EEG) em ratos jovens (21 dias). Em seguida foi verificado que a administração de LPS 3 horas antes da injeção intraestriatal de GA não alterou as convulsões, mas quando o LPS foi administrado 6 horas antes do GA, ele reduziu a latência e aumentou a duração das convulsões comportamentais e EEG induzidas pelo GA em ratos jovens. Observou-se também que injeção de LPS causou uma queda inicial na temperatura retal dos ratos jovens (até 2 horas), seguida de uma elevação na temperatura que iniciou em 3 horas e permaneceu alta até 6 horas após a injeção de LPS. Além disso, foi verificado que injeção de LPS 3 e 6 horas antes da injeção intraestriatal de GA causou um aumento nos níveis estriatais de IL-1β nos ratos jovens, sendo esse aumento estatisticamente maior em 6 do que em 3 horas. Também foi observado que o aumento nos níveis estriatais de IL-1β, causado pela administração de LPS, correlacionou-se positivamente com o tempo total de convulsões. Por fim, verificou-se que uso prévio do anticorpo da IL-1β preveniu a redução da latência e o aumento da duração das convulsões causadas pela administração de LPS 6 horas antes da injeção intraestriatal de GA nos ratos jovens. Assim, estes achados sugerem que a sinalização da IL-1β presente no processo inflamatório produzido pelo LPS contribui decisivamente para a hiperexcitabilidade neuronal e, consequentemente, para a redução da latência e o aumento da duração das convulsões induzidas pelo GA. Dessa maneira, tratamentos farmacológicos específicos que bloqueiam a superprodução ou as funções da IL-1β na GA-I, podem representar uma estratégia não convencional para o tratamento dessa patologia. Entretanto, estudos clínicos devem ser realizados a fim de avaliar a eficácia desse tratamento nos pacientes glutaricoacidêmicos que apresentam convulsões. Desde que os pacientes com GA-I apresentam outras alterações neurológicas importantes além das convulsões, como prejuízos cognitivos, a segunda hipótese a ser testada neste estudo foi verificar se o tratamento crônico com GA (5 μmol/g; s.c.; duas vezes por dia; do 5° ao 28° dia de vida) causaria déficit de memória espacial em ratos jovens, bem como se a inflamação produzida pelo LPS (2 mg/Kg; i.p.; uma vez por dia; do 25° ao 28° dia de vida) facilitaria o déficit cognitivo induzido pelo GA. Além disso, também foi objetivo avaliar o impacto desses tratamentos sobre possíveis alterações funcionais e estruturais no hipocampo desses animais. Inicialmente verificou-se que o tratamento crônico com GA, assim como os tratamentos com LPS e GA-LPS, causaram um déficit no aprendizado espacial dos ratos jovens. No entanto, foi observado que o tratamento com GA-LPS produziu um maior prejuízo na memória espacial comparado com os outros tratamentos. Em seguida foi observado que nenhum dos tratamentos alterou o peso ou a atividade locomotora/exploratória dos animais. Verificou-se também que o tratamento crônico com GA, assim como os tratamentos com LPS e GA-LPS, aumentaram os níveis hipocampais de IL-1β e TNF-α nos ratos jovens. Além disso, foi observado que tratamentos com GA, LPS e GA-LPS causaram uma redução no volume hipocampal total dos ratos jovens. Finalmente verificou-se que os tratamentos com GA, LPS e GA-LPS causaram uma redução na atividade da subunidade α1 da enzima Na+,K+-ATPase. Por outro lado, foi observado que os tratamentos com GA e LPS causaram um aumento na atividade das subunidades α2/3 da enzima. Assim, somente o tratamento com GA-LPS apresentou uma redução na atividade total da enzima Na+,K+-ATPase no hipocampo dos ratos jovens. Estes dados indicam que o prejuízo no aprendizado espacial observado nos ratos tratados com GA, LPS e GA-LPS parece estar relacionado a um aumento nos níveis de citocinas inflamatórias, a uma redução no volume hipocampal e a uma inibição na atividade da subunidade α1 da enzima Na+,K+-ATPase. No entanto, o maior prejuízo na memória espacial observado nos ratos tratados com GA-LPS ocorreu devido a inibição na atividade total da enzima Na+,K+-ATPase, que foi específica das isoformas α2/3, já que somente este grupo não apresentou resposta compensatória na atividade destas subunidades. Portanto, esta segunda parte do estudo demonstrou que o tratamento crônico com GA causou um déficit no aprendizado espacial de ratos jovens, e que a presença de um processo inflamatório potencializou o prejuízo na memória espacial induzida pelo GA sozinho. Assim, o entendimento dos mecanismos envolvidos nas convulsões e no déficit cognitivo observados nos paciente com GA-I frente a um processo inflamatório é importante para o desenvolvimento de novas terapias para o tratamento dessa patologia, bem como de outras doenças associadas à presença de mediadores inflamatórios.

Coordenação de Aperfeiçoamento de Pessoal de Nível Superior / Glutaric aciduria type I (GA-I) is an inborn error of metabolism (EIM) characterized biochemically by accumulation of glutaric acid (GA). The clinical manifestations are mainly neurological and develop during childhood. Among these changes, there are the seizures and cognitive deficits, which may be precipitated by infectious processes. Although growing evidence supports that inflammation and oxidative damage are both involved in learning impairment, it is not known whether inflammatory and oxidative stress markers facilitate GA-induced memory impairment. From this, the main objective of this study was to investigate the performance of rat pups chronically injected with GA and lipopolysaccharide (LPS) in spatial memory test on Barnes maze. To evaluate antioxidant defenses, cytokines levels, Na+, K+-ATPase activity, and hippocampal volume. Furthermore, we also evaluated wheter N-acetylcysteine (NAC) could improve these behavioral, biochemical or structural changes induced by GA and LPS administration. For this, the rat pups were injected with GA (5umol g of body weight-1, subcutaneously; twice per day; from 5th to 28th day of life), and were supplemented with NAC (150 mg/kg/day; intragastric gavage; for the same period). In order to mimic a severe infection state, LPS (2 mg/kg; E.coli 055 B5) or vehicle (saline 0.9%) was injected intraperitoneally, once per day, from 25th to 28th day of life.Oxidative stress biomarkers, antioxidant activity and hippocampal volume were assessed. In this study, GA caused spatial learning deficit in the Barnes maze, and that LPS potentiated the memory impairment induced by GA in rat pups. In addition, GA and LPS increased proinflammatory cytokine levels (TNF- and IL-1), and the co-administration of these compounds potentiated the increase of IL-1 levels but not TNF- levels in the hippocampus of this animals. Although GA and LPS administration increased TBARS (thiobarbituric acid-reactive substance) content, reduced antioxidant defenses and inhibited Na+,K+-ATPase activity (total and subunit α1), GA and LPS co-administration did not have additive effect on oxidative stress markers and Na+, K+ pump. The hippocampal volume did not change after GA or LPS administration. N-acetylcysteine protected against impairment of spatial learning and increase of cytokines levels induced by GA and LPS. The NAC also protected against deleterious effects induced by GA and LPS, as characterized by inhibition of Na+,K+-ATPase activity (total and subunit α1)and increase of TBARS content, as well as the reduction of antioxidant defenses(non protein thiols and glutathione content, superoxide dismutase and catalase activities).These results suggest that inflammatory and oxidative markers may underlie at least in part of the neuropathology of GA-I in this model. Pharmacological protection with NAC during encephalopatic crises could be considered as an adjuvant therapy to prevent hippocampal dysfunction and the progression of disease in children with GA-I. / A acidemia glutrárica do tipo I (AG-I) é um erro inato do metabolismo (EIM) caracterizada bioquimicamente pelo pelo acúmulo de ácido glutárico (AG). As manifestações clínicas são predominantemente neurológicas, e desenvolvem-se principalmente na infância. Entre essas alterações, as quais são precipitadas por processos infecciosos, pode-se citar o déficit cognitivo. Embora estudos recentes sugerem que a inflamação e o estresse oxidativo estão envolvidos no déficit cognitivo, não se sabe se os marcadores inflamatórios e oxidativos facilitam o prejuízo de memória após a administração de AG. A partir disso, o objetivo desta dissertação foi investigar o desempenho de ratos jovens injetados cronicamente com AG e lipopolissacarídeo (LPS) no teste de memória espacial no labirinto de Barnes. Além disso, foi avaliado os níveis das defesas antioxidantes, níveis de citocinas, atividade da enzima Na+, K+-ATPase e volume hipocampal. Como a N-acetilcisteína (NAC) possui propriedades antioxidantes e antiinflamatórias, foi testado se esse composto poderia melhorar as alterações comportamentais, bioquímicas e estruturais induzidas pela administração de AG e LPS. Para isso, os ratos jovens foram injetados com AG (5 μmol/g do peso corporal-1; subcutaneamente; duas vezes por dia; do 5º ao 28º dia de vida), e foram suplementados com NAC (150 mg/kg/dia; por gavagem; pelo mesmo período). A fim de mimetizar um estado infeccioso, LPS (2 mg/Kg: E. coli 055 B5) ou veículo (salina 0.9%) foi injetado intraperitonealmente uma vez por dia, do 25º ao 28º dia de vida. Nesse estudo, AG causou déficit de aprenizagem espacial no labirinto de Barnes, e o LPS potencializou esse prejuízo de memória induzido pelo AG nos ratos jovens. Em adição, a administração de AG e LPS aumentou os níveis de citocinas pró-inflamatórias (TNF- and IL-1), e a associação desses compostos potencializou o aumento dos níveis de IL-1, mas não de TNF-α no hipocampo dos animais. Embora a associação de AG e LPS tenha causado o aumento o conteúdo TBARS (espécies reativas ao ácido tiobarbitúrico), a redução das defesas antioxidantes e inibição da atividade da Na+,K+-ATPase (total e subunidade α1), a associação de AG e LPS não teve efeito aditivo nos marcadores de estresse oxidativo e na atividade da bomba de Na+ e K+. O volume hipocampal não foi alterado após a administração do AG e LPS. A N-acetilcisteína protegeu contra o prejuízo de aprendizagem espacial e aumento de citocinas inflamatórias induzido pelo AG e LPS. A NAC também protegeu contra os efeitos deletérios induzidos pelo AG e LPS, caracterizado pela inibição da atividade da Na+,K+-ATPase (total e subunidade α1) e aumento do conteúdo de TBARS, bem como a redução das defesas antioxidantes (tiós não-proteicos, conteúdo de glutationa, avitidade da superóxido dismutase e catalase). Esses resultados sugerem que marcadores inflamatórios e oxidativos podem estar envolvidos, em parte, na neuropatologia da AG-I neste modelo. Dessa forma, a proteção farmacológica com a NAC durante crises encefalopáticas pode ser considerada como uma terapia adjuvante para prevenir a disfunção hipocampal e a progressão da doença em crianças com AG-I.

Es ist davon auszugehen, dass Fehlfunktionen im Immunsystem mit der Ausprä-gung des Krankheitsbildes der aggressiven Parodontitis im Zusammenhang ste-hen. In dieser Arbeit sollen anhand klinischer, immunologischer und mikrobiologischer Untersuchungen ein immunologisches Risikoprofil bei Patienten mit aggressiver Parodontitis erschlossen, gegebenenfalls Unterschiede zur moderaten chronischen Parodontitis beleuchtet und explorativ Zusammenhänge zwischen immunologischen und mikrobiologischen Befunden eruiert werden. Es wurden geeignete Patienten und gesunde Probanden laut Ethikvotum rekrutiert. Die immunologischen Untersuchungen erfolgten an PBMCs unter Verwendung durchflusszytometrischer Methoden und mittels ELISpot-Assay. Mikrobiologische Untersuchungen subgingivaler Plaque wurden als klassische Kultur und 16S rRNA-Sequenzierung durchgeführt. Immundefekterkrankungen konnten bei allen Individuen ausgeschlossen werden. Im Gruppenvergleich wurde eine erhöhte Stimulierbarkeit der PBMCs von Patienten mit moderater chronischer Parodontitis bezüglich ihrer IL-1β-Freisetzung bei Inkubation mit LPS festgestellt. Des Weiteren wies diese Patientengruppe einen vergleichsweise höheren Anteil an Gedächtnis-B-Zellen auf. In der mikrobiologischen Untersuchung konnten bekannte parodontopathogene Spezies nachgewiesen und Prevotella denticola als bislang nicht explizit erwähntes Pathogen mit aggressiver Parodontitis assoziiert werden. Unsere Untersuchung weisen auf Zusammenhänge zwischen immunologischen und mikrobiologischen Befunde bezüglich einiger parodontopathogener Bakterien, wie Prevotella oralis, und Stimulierbarkeit der IL-1β-Freisetzung, B-Zelldifferenzierung und T-Zellverhältnis hin.

Besides exerting cytostatic or cytotoxic effects on tumor cells, some anti-cancer therapies (anthracyclines, oxaliplatin, X-Rays) could trigger an immunogenic cell death modality, releasing danger signals to alert immune system. We have shown that tumor-specific IFN- producing CD8+ T cells (Tc1) are mandatory for the success of chemotherapy to prevent tumor outgrowth. Priming of Tc1 response depends on IL-1β secretion by DC confronted with anthracycline-treated tumor cells releasing ATP. To identify the inflammatory components which link innate and cognate immune responses, we analyzed the influence of immunogenic chemotherapy on tumor microenvironment. We found an upregulated Th1- and Th17-related gene expression pattern in growth-retarded tumor after anthracycline treatment. By interfering with IFN- or IL-17A pathways, therapeutic effect of doxorubicin and oxaliplatin was abolished and dying tumor cell-based vaccine lost its efficacy to protect mice from live tumor cell rechallenge. Interestingly, we discovered that distinct subsets of  T lymphocytes (V4+ and V6+) colonized tumors shortly after chemotherapy, where they proliferated and became the dominant IL-17 producers within tumor beds. In three tumor models treated with chemotherapy or radiotherapy, a strong correlation between the presence of IL-17-producing  T ( T17) and IFN--producing CD8+ TIL (Tc1) was discovered. IL-17A signaling acts as upstream of IFN- since defect in IL-17RA led to complete loss of antigen specific Tc1 priming. The contribution of  T17 cells (V4+ and V6+) to chemotherapy is critical as V4/6-/- mice showed reduced sensitivity to chemotherapy and vaccination. Also, tumor infiltrating  T17 and Tc1 cells were reduced to basal level in this strain. IL-1β/IL-1R, but not IL-23/IL-23R, is pivotal for IL-17 production by  T cells and the success of chemotherapy. Importantly, adoptive transfer of  T cells could restore the efficacy of chemotherapy in IL-17A-/- mice and ameliorate the effect of chemotherapy in wild type host, provided that they retain the expression of IL-1R and IL-17A. Our research suggest a DC (IL-1β) →  T cells (IL-17) → Tc1 (IFN-) immune axis triggered by chemotherapy-induced dying tumor cells, which is critical for the favorable therapeutic response. To boost the immune system, we try to combine immunogenic chemotherapy with tumor vaccine in the presence of TLR3 agonist Poly (A:U). This sequential combined therapy, which we named VCT, could significantly retard tumor growth or even completely eradicate tumor and establish long-term protection against rechallenge in highly tumorigenic models. To dissect the effect of Poly (A:U) on immune system and that on TLR3 expressing-tumor cells, we performed VCT treatment in nude mice, TRIF-/- mice and with TRIF-silencing tumors. Interestingly, our results suggested that anti-tumor effect of VCT required T cells and intact TRIF signaling pathway at the level of the host and that of tumor cells. Poly (A:U) treatment could induce high level of CCL5 and CXCL10 production from tumor cells both in vitro and in vivo, which could negatively and positively influence the therapeutic outcome. By uncoupling the effect of CCL5 from that of CXCL10, the VCT treatment can be ameliorated. Our study emphasizes that both tumor and host derived inflammatory factors participate in regulating anti-tumor response. We also highlight that therapeutic application of TLR agonists can be optimized through regulating the profile of chemokines and their downstream signaling events.

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.

L’hypercholestérolémie familiale (FH) est un désordre lipidique associé aux maladies cardiovasculaires les plus fréquentes. La FH est causée par des mutations dans les gènes LDLR, APOB et PCSK9. Toutefois, chez 20% des patients souffrant de FH, aucune mutation dans ces gènes n'a été détectée et ceci suggère que d’autres gènes seraient à l’origine de la FH. Actuellement, le seul traitement de la FH est une thérapie aux statines. En général les statines sont bien tolérées, cependant, une monothérapie ne permet pas d’atteindre des niveaux thérapeutiques acceptables et dans bien des cas, une thérapie combinée devient nécessaire. De plus, l’intolérance aux statines est présente dans environ 12% des patients. Dans les trois dernières décennies, la survie des patients avec la FH a augmentée de façon notoire mais on observe aussi l’apparition d’une calcification vasculaire sévère chez certains d’entre eux. Il est donc primordial de développer des nouvelles approches thérapeutiques afin de prévenir ces complications tardives. Dans cette thèse doctorat, nous présentons l’étude d’une famille avec un phénotype de FH sévère non causé par des mutations dans les gènes LDLR, APOB et PCSK9. Par des études biochimiques et par séquençage d’ADN utilisant les technologies de nouvelle génération (NextGenSeq), nous avons découvert une mutation dans le gène de l’APOE (Leu167del). Ceci nous permet de proposer le gène codant pour l’APOE comme le 4e locus responsable de la FH (FH4). Par la suite, nous avons effectué deux études de cohortes chez les patients atteints de FH. Premièrement, dans l’étude JUPITER, nous avons démontré que la rosuvastatin augmente les niveaux sanguins de la protéine PCSK9 et ceci limiterait l’efficacité du traitement aux statines. Nous avons aussi étudié l’influence du mutant naturel R46L (perte de fonction de la PCSK9) dans la réponse aux statines. Deuxièmement, nous avons examiné les effets de la perte de fonction de la PCSK9 sur le profil cardiométabolique au sein d’une population pédiatrique. Nous avons déterminé que le génotype de l’APOE est déterminant dans ce profil cardiométabolique. Enfin, nous avons étudié la calcification vasculaire chez les patients atteints de FH. Cette calcification vasculaire progresse de façon indépendante des niveaux de cholestérol sérique et n’est pas associée aux anomalies de l’homéostasie du calcium. En utilisant des modèles murins, nous avons démontré que les souris Ldlr-/- et Tg(Pcsk9) développent des calcifications vasculaires semblables à celles observées chez l’homme. De plus, nous avons confirmé l’implication de la voie de signalisation LRP5/Wnt dans la pathophysiologie de la calcification artérielle. Avec une étude interventionnelle, nous avons trouvé que l’inhibition de l’interleukine 1β (IL-1β) diminue fortement l’apparition de calcifications vasculaire dans notre modèle murin. En conclusion, nos études ont permis l’identification d’un nouveau gène impliqué dans la FH, ont démontré aussi que les statines augmentent les niveaux sériques de PCSK9 et que la perte de fonction de la PCSK9 altère le profil cardiométabolique. Enfin, nous avons établi que la calcification vasculaire représente une complication tardive chez les patients atteints de FH et que, dans notre modèle murin, la calcification vasculaire peut être retardée par l’inhibition d’IL-1β. Ces découvertes peuvent avoir d’importantes répercussions cliniques chez l’humain. / Familial Hypercholesterolemia (FH) is the most common lipoprotein disorder associated with premature cardiovascular disease. Mutations in the LDLR, APOB and PCSK9 genes cause the FH phenotype, but in 20% of FH patients, no mutations in these genes are identified, suggesting that mutations in other genes cause FH. Treatment with statins has been the cornerstone of therapy. While statins are generally well tolerated, statin intolerance is found in approximately 12% of patients. Furthermore, statin use may not allow reaching LDL-C goals and combination therapy is often required. Nevertheless, survival of FH patients over the past 3 decades has improved significantly. As FH patients live longer, severe vascular calcifications have been described as a late complication in these patients. Given the increased survival rate and late complications, novel approaches and therapies are needed. In the present thesis we examined a kindred with a severe FH phenotype, where sequencing of candidate genes failed to identify a causal mutation. Through biochemical analysis and next-generation exome sequencing we report a mutation (Leu167del) within the APOE gene that identifies the 4th locus causing FH (FH4). Next, we performed two cohort-based studies. Firstly, in the JUPITER trial we report that 20mg rosuvastatin treatment increases PCSK9 levels by 30%, thereby possibly limiting the efficacy of statin therapy. Then we show the effect of a loss-of-function (LOF) mutation of PCSK9, p.R46L, on the response to rosuvastatin. Secondly, we report that two PCSK9 gene variants, p.R46L and insLEU, were more frequent in French Canadian individuals. We also report that the APOE genotype determine the metabolic risk profile in these mutations. Finally, we studied vascular calcifications in FH individuals. These calcifications appear to progress independently of cholesterol levels and are not associated with disturbances in calcium homeostasis. Using mouse models, we show that Ldlr-/- and Tg(Pcsk9) mice develop aortic calcifications similar to that observed in humans. Furthermore, the involvement of the LRP5/Wnt pathway in the pathogenesis of calcification is illustrated. In a proof-of-concept experiment, inhibiting the upstream pro-inflammatory cytokine IL-1β attenuates calcification in mice. In conclusion, we have contributed to the identification of a novel locus responsible for FH, reported the increase in PCSK9 levels with a statins treatment and the associated altered cardiometabolic profile in PCSK9 LOF. Finally, we demonstrated that vascular calcifications represent a severe complication of FH that can be prevented by inhibiting IL-1β in a mouse model. The latter novel approach may have an important translational application in human.

Une série d’études récentes chez l’homme et dans les modèles murins a conduit à la mise en évidence d’une réponse immune, qui met en jeu des cellules de l’immunité innée et de l’immunité adaptative dans le tissu adipeux (TA) obèse. Nous avons mis en évidence un dialogue pro-inflammatoire entre les macrophages et les LT CD4+ dans le TA humain obèse mettant en jeu l’IL-1β, l’IL-17 et l’IL-22. Le pourcentage de Th17 est positivement corrélé au % d’HbA1c. De plus, la sécrétion des cytokines impliquées dans cette boucle pro-inflammatoire diminuent après une perte de poids induite par la chirurgie bariatrique. Par la suite, nous avons montré que l’IL-1β et l’IL-17 induisent des programmes transcriptionnels pro-inflammatoires concordant dans trois types de cellules non-immunitaires du TA, les pré-adipocytes, les cellules endothéliales et les adipocytes, et diminuent les gènes du métabolisme dans les adipocytes. Les effets d’IL-1β sont nettement plus prononcés que ceux de l’IL-17. Le milieu conditionné de cellules immunitaires CD45+ reproduit les réponses pro-inflammatoire et catabolique induites par les cytokines recombinantes dans les adipocytes, et ces réponses sont inhibées après la neutralisation de l’IL-17 et l’IL-1β. Ces résultats démontrent une implication pathologique de l'IL-1β et de l'IL-17 dans les altérations du TA induites par l'obésité. / A series of recent studies in humans and mouse models has led to the detection of an immune response, which involves cells of the innate immunity and adaptive immunity in obese adipose tissue (AT).We highlighted a proinflammatory crosstalk between macrophages and CD4+ T cells in obese human AT involving IL-1β, IL-17 and IL-22. Percentage of Th17 is positively correlated with HbA1c %. Moreover, the secretion of the cytokines implicated in this proinflammatory loop decreased after weight loss induced by bariatric surgery.Subsequently, we have shown that IL-1β and IL-17 induce proinflammatory transcriptional programs in three types of non-immune cells of the TA, preadipocytes, endothelial cells and adipocytes , and decrease metabolism genes in adipocytes. IL-1β effects are much more pronounced than those of IL-17. The conditioned medium of CD45+ immune cells reproduces the pro-inflammatory and catabolic responses induced by the recombinant cytokines in adipocytes, and these responses are inhibited after the neutralization of IL-17 and IL-1β.These results demonstrate a pathological involvement of IL-1β and IL-17 in the AT dysfunction induced by obesity.

Aggregatibacter (Actinobacillus) actinomycetemcomitans is a bacterium mainly associated with aggressive forms of periodontitis. Among its virulence factors, a leukotoxin is suggested to play an important role in the pathogenicity. Periodontal infections with strains producing high levels of the leukotoxin are strongly associated with severe disease. Leukotoxin selectively kills human leukocytes and can disrupt the local defense mechanisms. Previous studies examining the role of the leukotoxin in host-parasite interactions have mainly focused on polymorphonuclear leukocytes (PMNs). In the inflamed periodontium, macrophages play a significant role in the regulation of the inflammatory reactions and the tissue breakdown and remodeling. Thus, the aim of this dissertation was to investigate death mechanisms of human macrophages exposed to leukotoxin. Human lymphocytes, PMNs, and monocytes/macrophages isolated from venous blood were exposed to purified leukotoxin or live A. actinomycetemcomitans strains producing variable levels or no leukotoxin. Different target cells were characterized by their expression of cell surface molecules. Cell death and viability were studied by examining cell membrane integrity and morphological alterations. Further, processes and cellular markers involved in apoptosis and necrosis were investigated. The expression and activation of pro-inflammatory cytokines of the leukotoxin-challenged leukocytes were examined at the mRNA and protein level. The biological activity of the secreted cytokines was investigated by testing the culture supernatants in a bone resorption assay. Additionally, different intracellular signaling pathways involved in the pro-inflammatory response from the macrophages were examined. Monocytes/macrophages were the most sensitive leukocytes for A. actinomycetemcomitans leukotoxin-induced lysis. This process in monocytes/ macrophages involved caspase-1 activation, and in addition, leukotoxin triggered abundant activation and secretion of IL-1β from these cells. The secreted IL-1β was mainly the 17 kDa bioactive protein and stimulated bone resorption. This activity could be blocked by an IL-1 receptor antagonist. When live bacteria were used, the A. actinomycetemcomitans-induced IL-1β secretion from human macrophages was mainly caused by the leukotoxin. Closer examination of the macrophages exposed to leukotoxin revealed that the induced cell death proceeded through a process that differed from classical apoptosis and necrosis. Interestingly, this process resembled a newly discovered death mechanism termed pyroptosis. The extensive leukotoxin induced IL-1β secretion did not correlate to increased levels of mRNA for IL-1β. It was mainly mediated by caspase-1 activation, since blocking it by a specific inhibitor also abolished the secretion of IL-1β. A similar pattern, but at much lower level, was seen for IL-18. In conclusion, these results show that A. actinomycetemcomitans leukotoxin induces a death process in human macrophages leading to a specific and excessive pro-inflammatory response. Our results indicate that this novel virulence mechanism of leukotoxin may play an important role in the pathogenic potential of A. actinomycetemcomitans.

L’hypercholestérolémie familiale (FH) est un désordre lipidique associé aux maladies cardiovasculaires les plus fréquentes. La FH est causée par des mutations dans les gènes LDLR, APOB et PCSK9. Toutefois, chez 20% des patients souffrant de FH, aucune mutation dans ces gènes n'a été détectée et ceci suggère que d’autres gènes seraient à l’origine de la FH. Actuellement, le seul traitement de la FH est une thérapie aux statines. En général les statines sont bien tolérées, cependant, une monothérapie ne permet pas d’atteindre des niveaux thérapeutiques acceptables et dans bien des cas, une thérapie combinée devient nécessaire. De plus, l’intolérance aux statines est présente dans environ 12% des patients. Dans les trois dernières décennies, la survie des patients avec la FH a augmentée de façon notoire mais on observe aussi l’apparition d’une calcification vasculaire sévère chez certains d’entre eux. Il est donc primordial de développer des nouvelles approches thérapeutiques afin de prévenir ces complications tardives. Dans cette thèse doctorat, nous présentons l’étude d’une famille avec un phénotype de FH sévère non causé par des mutations dans les gènes LDLR, APOB et PCSK9. Par des études biochimiques et par séquençage d’ADN utilisant les technologies de nouvelle génération (NextGenSeq), nous avons découvert une mutation dans le gène de l’APOE (Leu167del). Ceci nous permet de proposer le gène codant pour l’APOE comme le 4e locus responsable de la FH (FH4). Par la suite, nous avons effectué deux études de cohortes chez les patients atteints de FH. Premièrement, dans l’étude JUPITER, nous avons démontré que la rosuvastatin augmente les niveaux sanguins de la protéine PCSK9 et ceci limiterait l’efficacité du traitement aux statines. Nous avons aussi étudié l’influence du mutant naturel R46L (perte de fonction de la PCSK9) dans la réponse aux statines. Deuxièmement, nous avons examiné les effets de la perte de fonction de la PCSK9 sur le profil cardiométabolique au sein d’une population pédiatrique. Nous avons déterminé que le génotype de l’APOE est déterminant dans ce profil cardiométabolique. Enfin, nous avons étudié la calcification vasculaire chez les patients atteints de FH. Cette calcification vasculaire progresse de façon indépendante des niveaux de cholestérol sérique et n’est pas associée aux anomalies de l’homéostasie du calcium. En utilisant des modèles murins, nous avons démontré que les souris Ldlr-/- et Tg(Pcsk9) développent des calcifications vasculaires semblables à celles observées chez l’homme. De plus, nous avons confirmé l’implication de la voie de signalisation LRP5/Wnt dans la pathophysiologie de la calcification artérielle. Avec une étude interventionnelle, nous avons trouvé que l’inhibition de l’interleukine 1β (IL-1β) diminue fortement l’apparition de calcifications vasculaire dans notre modèle murin. En conclusion, nos études ont permis l’identification d’un nouveau gène impliqué dans la FH, ont démontré aussi que les statines augmentent les niveaux sériques de PCSK9 et que la perte de fonction de la PCSK9 altère le profil cardiométabolique. Enfin, nous avons établi que la calcification vasculaire représente une complication tardive chez les patients atteints de FH et que, dans notre modèle murin, la calcification vasculaire peut être retardée par l’inhibition d’IL-1β. Ces découvertes peuvent avoir d’importantes répercussions cliniques chez l’humain. / Familial Hypercholesterolemia (FH) is the most common lipoprotein disorder associated with premature cardiovascular disease. Mutations in the LDLR, APOB and PCSK9 genes cause the FH phenotype, but in 20% of FH patients, no mutations in these genes are identified, suggesting that mutations in other genes cause FH. Treatment with statins has been the cornerstone of therapy. While statins are generally well tolerated, statin intolerance is found in approximately 12% of patients. Furthermore, statin use may not allow reaching LDL-C goals and combination therapy is often required. Nevertheless, survival of FH patients over the past 3 decades has improved significantly. As FH patients live longer, severe vascular calcifications have been described as a late complication in these patients. Given the increased survival rate and late complications, novel approaches and therapies are needed. In the present thesis we examined a kindred with a severe FH phenotype, where sequencing of candidate genes failed to identify a causal mutation. Through biochemical analysis and next-generation exome sequencing we report a mutation (Leu167del) within the APOE gene that identifies the 4th locus causing FH (FH4). Next, we performed two cohort-based studies. Firstly, in the JUPITER trial we report that 20mg rosuvastatin treatment increases PCSK9 levels by 30%, thereby possibly limiting the efficacy of statin therapy. Then we show the effect of a loss-of-function (LOF) mutation of PCSK9, p.R46L, on the response to rosuvastatin. Secondly, we report that two PCSK9 gene variants, p.R46L and insLEU, were more frequent in French Canadian individuals. We also report that the APOE genotype determine the metabolic risk profile in these mutations. Finally, we studied vascular calcifications in FH individuals. These calcifications appear to progress independently of cholesterol levels and are not associated with disturbances in calcium homeostasis. Using mouse models, we show that Ldlr-/- and Tg(Pcsk9) mice develop aortic calcifications similar to that observed in humans. Furthermore, the involvement of the LRP5/Wnt pathway in the pathogenesis of calcification is illustrated. In a proof-of-concept experiment, inhibiting the upstream pro-inflammatory cytokine IL-1β attenuates calcification in mice. In conclusion, we have contributed to the identification of a novel locus responsible for FH, reported the increase in PCSK9 levels with a statins treatment and the associated altered cardiometabolic profile in PCSK9 LOF. Finally, we demonstrated that vascular calcifications represent a severe complication of FH that can be prevented by inhibiting IL-1β in a mouse model. The latter novel approach may have an important translational application in human.

Traumatic Brain Injury (TBI) mainly affects young persons in traffic accidents and the elderly in fall accidents. Improvements in the clinical management have significantly improved the outcome following TBI but survivors still suffer from depression, memory problems, personality changes, epilepsy and fatigue. The initial injury starts a series of events that give rise to a secondary injury process and despite several clinical trials there is no drug available for clinical use that targets secondary brain injury mechanisms. Some recovery of function is seen during the first months following injury but is usually limited and there are no drugs that stimulate the recovery of lost function. Some of the recovery is attributed to plasticity, the brains ability to adapt to new circumstances, and enhancing plasticity via increased axonal growth has the potential to partly restore lost function. In this thesis mice were subjected to the controlled cortical impact model of TBI and functional outcome was evaluated using Morris water maze, the cylinder test and the rotarod. Brain tissue loss was measured in all Papers but the additional histological analyses differ among the Papers. Attempts to increase axonal growth were made by interfering with Nogo receptor function in Paper I and by conditional knockout of ephA4 in Paper II. Contrary to the hypothesis cognition was impaired in Paper I but otherwise no effects of treatment were detected in Paper I and II. Much is still unknown about plasticity and despite the discouraging results of Papers I and II this treatment approach is still worth further exploration. It is firmly established that TBI results in an inflammatory response and some aspects of it may damage brain tissue. In Papers III and IV the inflammatory response was attenuated using an IL-1β directed antibody which resulted in reduced tissue loss and edema while improving cognitive function. The results from Papers III and IV are encouraging and the possibility to find a treatment based on IL-1β inhibition appears promising.