Actinobacillus actinomycetemcomitans is present in elevated proportions and numbers in dental bacterial biofilms of patients with localized aggressive periodontitis. This variant of periodontal disease, occurring in adolescents and young adults, is characterized by rapid and severe destruction of the connective tissues and bone supporting the teeth, eventually culminating in tooth loss. The cytolethal distending toxin (Cdt) is a newly discovered bacterial protein toxin, uniquely present in A. actinomycetemcomitans among all known to-date oral bacterial species. The Cdt has the capacity to inhibit mammalian cell growth, but its putative role in the pathogenesis of the disease is unclear. The aim of this in vitro work has been to study the effects of A. actinomycetemcomitans on periodontal connective tissue cell cultures, and to evaluate the possible involvement of its Cdt. A. actinomycetemcomitans inhibited the proliferation of gingival and periodontal ligament fibroblasts, as a result of a combined arrest at the G1 and G2/M phases of the cell cycle. This growth inhibition was non-lethal and the cells remained metabolically active, although their DNA synthesis was reduced. The intoxicated cells exhibited increased size and irregular structure, characterized by distension and elongation. This cellular enlargement occurred in both G1 and G2/M phase arrested cells. The Cdt of A. actinomycetemcomitans was responsible for the observed growth inhibition, as well as the concomitant morphological alterations. The possible induction of inflammatory cytokines related to bone resorption was investigated in response to A. actinomycetemcomitans, and the involvement of Cdt was evaluated. Extensive focus was given to the study of receptor activator of NF-κB ligand (RANKL) expression, a membrane-bound ligand that signals osteoclast progenitors to differentiate and fuse into mature osteoclasts, activating bone resorption. It was demonstrated that A. actinomycetemcomitans induced RANKL mRNA and protein expression in the cells studied, but did not affect the expression of its decoy receptor, osteoprotegerin. This induction was solely attributed to its Cdt, as demonstrated by the use of a cdt-knockout A. actinomycetemcomitans strain, purified recombinant Cdt, and antibodies blocking the Cdt. In addition, this event was not mediated by pro-inflammatory cytokines known to stimulate RANKL. Interleukin-6 mRNA and protein expression were also enhanced by A. actinomycetemcomitans, but Cdt had limited involvement in this enhancement. In conclusion, two distinct mechanisms by which A. actinomycetemcomitans Cdt may be involved in the pathogenesis of localized aggressive periodontitis are proposed. Firstly, the growth arrest of the resident fibroblasts may impair the physiological connective tissue remodelling equilibrium and lead to connective tissue attachment loss. Secondly, the induction of RANKL by these cells, residing in the proximity of the alveolar bone, may locally stimulate osteoclastogenesis and promote alveolar bone resorption. This work also provides further insights to the understanding of Cdt mechanisms of action, contributing to the global characterization of the toxin’s virulence.

Les neutrophiles sont généralement considérés résistants aux glucocorticoïdes. Cependant, peu d’études comparant l’effet de ces drogues sur les neutrophiles et les autres leucocytes sanguins (monocytes, lymphocytes et éosinophiles) ont été rapportées. Dans notre étude, nous avons évalué la réponse aux glucocorticoïdes de ces deux populations cellulaires chez le cheval et l’homme. Les cellules, préalablement isolées du sang de 6 chevaux et 4 sujets humains sains, ont été incubées pendant 5 h en présence de lipopolysaccharide (LPS; 100 ng/mL) seul ou combiné avec de l’hydrocortisone, de la prednisolone ou de la dexaméthasone (10-8M et 10-6M). L’expression d’ARNm pour l’IL-1β, le TNF-α, l’IL-8, la glutamine synthétase et le récepteur α des glucocorticoïdes (GR-α) a été quantifiée par qPCR. Les neutrophiles équins ont également été incubés pendant 20 h en présence de ces 3 glucocorticoïdes et la survie cellulaire a été évaluée par cytométrie de flux et microscopie optique. Nous avons démontré que les glucocorticoïdes inhibaient l’expression des gènes pro-inflammatoires induite par le LPS pour les deux populations cellulaires chez les deux espèces étudiées. L’expression de la glutamine synthétase était également significativement augmentée par les glucocorticoïdes chez les neutrophiles et les autres leucocytes sanguins équins. De manière générale, l’intensité de la réponse aux glucocorticoïdes s’est avérée similaire dans les 2 populations leucocytaires et chez les deux espèces. Les glucocorticoïdes augmentaient également la survie des neutrophiles équins, phénomène également rapporté dans d’autres espèces. Ainsi, les glucococorticoïdes exercent des effets d’intensité comparable sur les neutrophiles et les autres leucocytes sanguins. Nous spéculons que la faible réponse à la corticothérapie observée lors de maladies inflammatoires chroniques neutrophiliques comme l’asthme sévère ou la Maladie Pulmonaire Obstructive Chronique (MPOC) ne s’explique pas par une corticorésistance intrinsèque des neutrophiles. / Neutrophils are generally considered resistant to glucocorticoids compared to other inflammatory cells. However, there are few studies comparing the effects of glucocorticoids in neutrophils and those of other blood leukocytes (monocytes, lymphocytes and eosinophils). In our study, we assessed glucocorticoid-responsiveness in equine and human peripheral blood neutrophils and in neutrophil-depleted leukocytes. Cells were isolated from 6 healthy horses and 4 human healthy subjects. They were incubated for 5 h with or without lipopolysaccharide (LPS; 100 ng/mL) alone or combined with hydrocortisone, prednisolone or dexamethasone (10-8M and 10-6M). IL-1β, TNF-α, IL-8, glutamine synthetase and Glucocorticoid Receptor α (GR-α) mRNA expression was quantified by qPCR. Equine neutrophils were also incubated for 20 h with or without the three glucocorticoids and cell survival was assessed by flow cytometry and light microscopy. We found that glucocorticoids down-regulated LPS-induced pro-inflammatory mRNA expression in both cell populations and species. These drugs also significantly increased glutamine synthetase gene expression in both equine cell populations. The magnitude of glucocorticoid response was generally similar in both cell populations and species. As reported in other species, glucocorticoids significantly increase the survival in equine neutrophils. Based on these results, it appears that glucocorticoids exert effects of similar magnitude on neutrophils and on other blood leukocytes. We speculate that the poor response to glucocorticoids observed in some chronic neutrophilic human diseases such as severe asthma or Chronic Obstructive Pulmonary Disease (COPD) is not explained by an inherent attenuated response of neutrophils to these drugs.

Le syndrome d'ischémie-reperfusion (IR), inhérent à la transplantation rénale, est caractérisé par un infiltrat leucocytaire important et des lésions tissulaires graves dont les signaux initiateurs restent à ce jour peu décrits. Postulant que la libération d'alarmines par les cellules en nécrose est décisive dans ce processus, l'objectif principal du présent travail a été d'étudier la contribution de l'alarmine IL-33 dans la genèse des lésions tissulaires dans un modèle murin d'IR rénale. Nos résultats montrent que l'IL-33 est rapidement libérée du rein après IR comme protéine circulante, dès une heure de reperfusion. Les souris IL-33gt/gt, déficientes en IL-33, sont moins sensibles aux lésions induites par l’IR, comme l'attestent le maintien de la fonction rénale et des lésions histologiques atténuées avec un recrutement de polynucléaires neutrophiles (PNN) diminué par rapport aux souris contrôles. Ceci est associé à la perte du recrutement de cellules iNKT productrices d'IFN-γ/IL-17A. Parallèlement, les souris Jα18KO, déficientes en cellules iNKT et protégées contre les lésions d'IR, possèdent également des niveaux élevés d'IL-33 circulante. Nous proposons donc que l'IL-33 endogène contribue aux lésions d'IR en favorisant le recrutement de cellules iNKT, conduisant ainsi à un recrutement amplifié de PNN au niveau du rein lésé. Notre étude, en identifiant l'alarmine IL-33 comme un médiateur précoce de la réponse immunitaire innée induite par l'IR rénale, mettant en jeu les cellules iNKT, contribue à la compréhension des mécanismes impliqués dans la genèse des lésions associées à la greffe rénale et permet de proposer de nouvelles stratégies thérapeutiques. / Ischemia-reperfusion (IR) injury in renal transplantation is characterized by leukocyte infiltration and tissue damage. However, the signals that initiate these events remain poorly understood. Assuming that alarmin release by necrotic cells during IRI is critical, the main objective of the present study was to investigate the role of alarmin IL-33 in kidney injury using a mouse model of renal IR. We observed release of IL-33 shortly after kidney IR concomitantly with an increase in plasma levels of IL-33 within one hour of reperfusion. IL-33 deficient mice (IL-33gt/gt) exhibited reduced renal IR-induced injury, as attested by function preservation, reduced histological change and attenuation of neutrophil recruitment compared to control mice. This was associated with the loss of IFN-γ/IL-17A-producing iNKT cell recruitment. In the meantime, iNKT cell-deficient (Jα18KO) mice, also protected against IRI, have increased levels of circulating IL-33.These findings lead us to propose that endogenous IL-33 contributes to kidney IRI by promoting iNKT cell recruitment and cytokine production, thereby promoting amplified neutrophil recruitment to the injured kidney. The present study identifies the nuclear alarmin interleukin (IL)-33 as an important and early mediator of innate immune response, involving iNKT cells, following experimental kidney ischemia-reperfusion in mice. Our findings contribute to a better understanding of IR-induced injury and may lead to new therapeutic insights into renal-induced injury associated with renal transplantation.

Coordenação de Aperfeiçoamento de Pessoal de Nível Superior - CAPES / Pesticides are widely used in agriculture and public health for pest control and prevention. The indiscriminate use of these compounds can trigger environmental contamination by pesticides and increase the risk of human exposure. Human exposure to pesticides can occur directly, through occupational activity, or indirectly, through the environment and food. Chronic exposure to pesticides may result in neurological, reproductive, teratogenic and immunological disorders. Mancozeb, chlorothalonil and thiophanate methyl are fungicides widely used in the world. Despite their characteristics of low acute toxicity and low persistence in the environment, in vitro and in vivo studies demonstrate the cytotoxic effects of these compounds. However, the immunological effects that these pesticides can trigger are unexplored. Therefore, the objective of this study was to evaluate in vitro the pro-inflammatory and oxidative effects of mancozeb, chlorothalonil and thiophanate methyl pesticides. RAW 264.7 (ATCC® TIB-71™) macrophages were exposed to different concentrations (0.1-100 μg/mL) of each pesticide for 72 hours, and maintained in 5% CO2 atmosphere at 37°C. The pesticides were dissolved in dimethylsulfoxide, which was used as negative control. Phytohemagglutinin was used as positive control for inflammatory activation. The evaluation of cell proliferation, oxidative metabolism and pro-inflammatory cytokines (IL-1β, IL-6, TNF-α and IFN-γ), anti-inflammatory cytokine (IL-10) and caspases (Casp1, Casp3, Casp8) levels were performed by fluorimetric and molecular tests. The results showed a significant pro-inflammatory effect of mancozeb, chlorothalonil and thiophanate methyl pesticides at respective concentrations of 1, 3 and 100 μg/mL, with increase in cell proliferation and pro-inflammatory cytokines and caspases levels. However, the oxidative effect was only observed in macrophages exposed to chlorothalonil at 3 μg/mL. Thus, in these analysis conditions, the studied pesticides acted by activation of the immune system. The results found contribute to better understanding of immunological effects caused by exposure to these pesticides. / Os pesticidas são amplamente utilizados na agricultura e na saúde pública para o controle e prevenção de pragas. O uso indiscriminado desses compostos pode desencadear a contaminação ambiental por agrotóxicos e aumentar o risco de exposição dos seres humanos. A exposição aos agrotóxicos pelos humanos pode ocorrer de forma direta, através de atividade ocupacional, ou de forma indireta, pelo ambiente e pela alimentação. A exposição crônica aos pesticidas pode resultar em distúrbios neurológicos, reprodutivos, teratogênicos e imunológicos. Os pesticidas mancozebe, clorotalonil e tiofanato metílico são fungicidas amplamente utilizados no mundo. Apesar de suas características de baixa toxicidade aguda e baixa persistência no ambiente, estudos in vitro e in vivo demonstram os efeitos citotóxicos desses compostos. Entretanto, os efeitos imunológicos que esses pesticidas podem desencadear são pouco explorados. Diante disso, o objetivo deste estudo foi avaliar in vitro o efeito pró-inflamatório e oxidativo dos pesticidas mancozebe, clorotalonil e tiofanato metílico. Os macrófagos RAW 264.7 (ATCC® TIB-71™) foram expostos a diferentes concentrações (0,1 – 100 μg/mL) de cada pesticida por 72 horas, sendo mantidos em atmosfera com 5% CO2 a 37oC. Os pesticidas foram dissolvidos em dimetilsulfóxido, o qual foi utilizado como controle negativo. Como controle positivo para a ativação inflamatória, utilizou-se a fitohemaglutinina. A avaliação da proliferação celular, do metabolismo oxidativo e dos níveis das citocinas pró-inflamatórias (IL-1β, IL-6, TNF-α e IFN-γ), da citocina anti-inflamatória (IL-10) e das caspases (Casp1, Casp3, Casp8) foi realizada por testes fluorimétricos e moleculares. Os resultados obtidos demonstraram efeito pró-inflamatório significativo dos pesticidas mancozebe, clorotalonil e tiofanato metílico nas respectivas concentrações de 1, 3 e 100 μg/mL, ocorrendo aumento da proliferação celular e dos níveis de citocinas pró-inflamatórias e caspases. Entretanto, o efeito oxidativo somente foi observado nos macrófagos expostos ao clorotalonil na concentração de 3 μg/mL. Assim, nessas condições de análise, os pesticidas estudados atuam ativando o sistema imune. Os resultados encontrados contribuem para a melhor compreensão dos efeitos imunológicos que a exposição a estes pesticidas pode desencadear.

CoordenaÃÃo de AperfeiÃoamento de Pessoal de NÃvel Superior / This work as main objective to evaluate the eletrogenic effect in preparations of Ãleum of rabbit fixes in chambers of Ãssing, in presence of supernatant of macrophages (S.MfS), stimulated with microcistin-LR (MCLR), of Microcystis aeruginosa. S.MfS estimulated with MCLR (3,2.10-7M; 9,6.10-7M e 3,2.10-6M), produces effect secretion, of the form dose-dependent; being that short circuit current (Isc), the secular chain variation (t) can be described for an equation of the type Isc = a . ekt for a correlation coefficient r = 0,9988 and Iscmaximo = 128,16  14,54 ÂA . cm-2. Later, was observed that the metabolic processes associatesâ geneses of the FSI, from stimulated macrophages, require the participation of a protein G, sensible pertussis active toxin. It was also verified that inhibing of protÃic synthesis, proteases, phosfolipase A2, ciclooxigenases, lipoxigenases, synthesis TNF-a, and antagonists of the PAF, they had reduced the FSI synthesis. With the application of monoclonal antibodies, it was verified that interleukin-b (IL-1b), was the main FSI; as also, that macrophages stimulated with MCLR, in the concentrations above, produced IL-1b and TNF-a, of form dose-dependent. The pay-treatment of the ileum mucosal with bumetanide, indomethacin, tetrodotoxin and HOE, disclosed that the secretory effect, by means of stimulated action of the S.MfS with MCLR is dependent of the secretion of ions chloride, with the participation of PAF, prostaglandins and mediators of the enteric nervous system. With this effect, it associates reduction of the transepitelial resistance (Rte), also mediated for prostaglandins, TNF-a, and indirectly IL-1b. The analysis of the coefficient of Hurst, disclosed that these effect had not occurred of random form, but had involved significant alterations in the kinetic parameters of the eletrogÃnic effect, to the level of the ileum mucosal. Macrophages stimulated for MCLR, produces and liberate more TNF-a of that IL-1b, to the Constant taxes, being this a linked characteristic to the type of employed stimulation. It is concluded, therefore, that MCLR stimulates macrophages to produce substances that can act as intestinal secretion factor, to the level of the ileum mucosal, involving chloride canals, reduction of the Rte, requesting for this, the participation of pro-inflammatory mediators and the enteric nervous system. / Este trabalho teve-se como principal objetivo, avaliar os efeitos eletrogÃnicos em preparaÃÃes de Ãleo de coelho fixadas em cÃmaras de Ãssing, em presenÃa de sobrenadante de macrÃfagos (S.MfS) estimulados com microcistina-LR MCLR de Microcystis aeruginosa. S.MfS estimulados com MCLR (3,2.10-7M; 9,6.10-7M e 3,2.10-6M), produz, de forma dose-dependente; sendo que a variaÃÃo temporal (t) da corrente de curto-circuito (Isc), pode ser descrita por uma equaÃÃo do tipo Isc = a . ekt; para um coeficiente de correlaÃÃo r = 0,9988 e Iscmaximo = 128,16  14,54 ÂÂcm-2. Posteriormente, observou-se que os processos metabÃlicos associados à gÃnese do fator deâ secreÃÃo intestinalâ (FSI), a partir de macrÃfagos estimulados, requer a participaÃÃo de uma proteÃna G sensÃvel à toxina pertusis ativa. Verificou-se tambÃm que inibidores de sÃntese protÃica, proteases, fosfolipase A2, cicloxigenases, lipoxigenases,; sÃntese de TNF-a e antagonista do PAF, reduziram a sÃntese de FSI. Com o emprego de anticorpos monoclonais, verificou-se que IL-1b era o principal FSI; como tambÃm, que macrÃfagos estimulados com MCLR, nas concentraÃÃes acima, reduziu IL-1b e TNF-a, de forma dose-dependente. O prÃ-tratamento da mucosa ileal com bumetanida, indometacina, tetrodotoxina e HOE, revelou que o efeito secretÃrio mediante aÃÃo do S.MfS estimulados com MCLR, à dependente da secreÃÃo de Ãons cloreto, com a participaÃÃo de PAF, prostaglandinas e mediadores do sistema nervoso entÃrico. A este efeito associa-se a diminuiÃÃo da resistÃncia transepitelial (Rte), tambÃm mediada por, prostaglandinas, PAF, TNF-a e, indiretamente, IL-1b. A anÃlise do coeficiente de Hurst (H), revelou que estes efeitos nÃo ocorreram ao acaso, mas envolveram alteraÃÃes significativas nos parÃmetros cinÃticos dos efeitos eletrogÃnicos, ao nÃvel da mucosa ileal. MacrÃfagos estimulados com MCLR produz e libera mais TNFa do que IL-1b, à taxas constantes ; sendo esta uma caracterÃstica atrelada ao tipo de estÃmulo empregado. Conclui-se, portanto, que MCLR estimula macrÃfagos a produzir substÃncias que podem atuar como fator de âsecreÃÃo intestinalâ ao nÃvel da mucosa ileal, envolvendo canais de cloreto, diminuiÃÃo de Rte; requisitando para isto, a participaÃÃo de mediadores prÃ-inflamatÃrios e do sistema nervoso entÃrico.

Dans les patients infectés par HBV, les thérapies avec les analogues de nucléos(t)ides (NAs) ou l'interféron α (IFNα) restent inefficaces pour éradiquer l'infection, à cause d'une forme persistante d'HBV, appelée l'ADN circulaire covalent clos (ADNccc), organisé comme un mini-chromosome. Notre but a été de revisiter l'activité anti-HBV d'un panel de cytokines in vitro en utilisant des hépatocytes non transformés, afin d'identifier de nouvelles options immunothérapeutiques. Parmi toutes les molécules testées, l'IFNβ, l'IFNγ, les IFNλ, le TNFα, l'IL-6, l'IL-1β et le ténofovir (ce dernier utilisé comme contrôle positif) ont montré un effet suppresseur sur la réplication d'HBV aussi fort et parfois plus fort que l'IFNα. La cytokine ayant l'effet le plus élevé sur l'ADN total d'HBV (EC50 ≈ 25 pg/mL), sans cytotoxicité, était l'interleukine-1β (IL-1β), qui est naturellement produite par les cellules de Kupffer (KC), les macrophages du foie. De façon importante, les ARNs totaux d'HBV et l'antigène sécrété HBeAg, mais pas HBsAg, ni l'ADNccc, sont fortement diminués par l'IL-1β. Nous avons donc émis l'hypothèse selon laquelle des promoteurs viraux spécifiques su l'ADNccc pourraient être inhibés, même si l'ADNccc n'est pas dégradé. Ensuite, nous avons étudié le mécanisme de l'activité antivirale de l'IL-1β. Nous avons montré que tous les promoteurs d'HBV sembleraient être inhibés par l'IL-1β. En parallèle, nous avons vérifié que l'IL-1β pouvait activer le promoteur de NF-κB, dont la fonction de transcription a été confirmée. Grâce à cette étude, l'IL-1β a été montré comme ayant un effet antiviral très efficace contre HBV in vitro, par l'intermédiaire de la fixation de NF-κB sur l'ADNccc / In HBV-infected patients, therapies with nucleos(t)ide analogues (NAs) or interferon α (IFNα) remain ineffective in eradicating the infection, because of a persistent form of HBV DNA, namely the covalently closed circular DNA (cccDNA), which is organized as a minichromosome. Our aim was to revisit the anti-HBV activity of a panel of IFNs and pro-inflammatory cytokines in vitro using nontransformed cultured hepatocytes of HBV infection, to identify new immunotherapeutic options. Amongst all molecules tested, IFNβ, IFNγ, IFNλ, TNFα, IL-6, IL-1β and tenofovir showed a suppressive effect on HBV replication at least as strong as, but sometimes stronger than IFNα. The cytokine showing the highest effect on intracellular total HBV DNA without any cytotoxicity, was interleukin-1β (IL-1β), which is naturally produced by Kupffer cells (KC), representing the macrophages of the liver. Importantly, total HBV RNAs and secreted HBeAg, but nor HBsAg, neither cccDNA, were strongly decreased. Thus, we hypothesized that even if cccDNA was not degraded, specific viral promoters on cccDNA could be silenced. Then, we investigated the mechanism of IL-1β antiviral activity. We have shown that all HBV promoters were early inhibited by IL-1β. In the meantime, we have verified that IL-1β can induce nuclear Translocation and expression of NF-κB. We also checked NF-κB functionality. Thanks to this study, IL-1β has been found to have very potent antiviral effect against HBV in vitro, through the binding of NF-κB on cccDNA

Les propriétés exceptionnelles des nanotubes de carbone (CNT) attirent de nombreux industriels dans les domaines de la microélectronique, des matériaux ou de la nanomédecine. Néanmoins, le risque sanitaire lié à ce nanomatériau reste encore mal compris. Des profils toxicologiques différents, dépendant des caractéristiques physico-Chimiques des CNT, ont été mis en évidence. Une approche « safer by design » est proposée, afin d’identifier les paramètres pouvant, dès la conception des CNT, pour limiter le risque sanitaire. Dans ce contexte, cette thèse avait pour objectif d’étudier l’impact sur la réponse in vitro d’une lignée de macrophages murins (RAW 264.7) de deux traitements de post-Production de CNT : la fonctionnalisation acide et le recuit haute température.Les groupements acides en surface des CNT fonctionnalisés ont entrainé une augmentation de la réponse pro-Inflammatoire sans influencer significativement la cytotoxicité. D’un autre côté, la fonctionnalisation acide, principalement par l’élimination des impuretés métalliques, a permis de diminuer le stress oxydant. Les CNT recuits à haute température étaient à l’origine d’une réponse pro-Inflammatoire plus importante que les CNT bruts, confirmant lasensibilité de cette réponse biologique à la chimie de surface. En revanche, le recuit n’a pas diminué significativement le stress oxydant malgré la purification des CNT, suggérant l’importance des défauts de structure sur cette réponse biologique. La fonctionnalisation acide de nano-Graphite et de noir de carbone a eu un impact similaire à celle des CNT sur l’activité biologique des macrophages. La comparaison de ces trois nanomatériaux fonctionnalisés semble s’accorder avec le paradigme mettant en exergue la toxicité spécifique des fibres et des plaquettes. Enfin, afin de compléter ces résultats, des études exploratoires sur les interférences entre les tests de toxicité et les CNT, ainsi que sur le stress oxydant, ont été conduites. / Due to their exceptional properties, carbon nanotubes (CNT) have aroused a huge interest among in industrial fields such as microelectronics, material science and nanomedicine. Nevertheless, the health impacts of this nanomaterial still remain not well understood. The first toxicological studies pointed out that there is no unique response regarding the healthimpact of the CNT, but different toxicological profiles according to their various physicochemical properties. A safer by design approach is thus proposed to identify the parameters decreasing from their production the CNT biological impacts. In this context, this work aimed at studying the impact on the in vitro response from a macrophage cell line (RAW 264.7) of two post-Production treatments: acid functionalization and high temperature annealing.Surface acid groups from functionalized CNT enhanced the pro-Inflammatory response although the cytotoxicity remained stable. On the other hand, acid functionalization, through the elimination of metallic impurities, significantly decreased the oxidative stress. Annealed CNT increased the pro-Inflammatory response compared to the pristine CNT. It thus confirmed the sensitivity of this response for the changes in surface chemistry. However, the high temperature annealing did not influence the oxidative stress, despite of the CNT purification. It suggested that structural defects are also of importance for this response. Besides, the acid functionalization of nano-Graphite and carbon black displayed trends in the macrophage response similar to the acid functionalization of CNT. The comparison of these three carbon-Based nanomaterials seemed to conform to the fibre and platelets paradigm. Eventually, exploratory studies have also been conducted on the interferences between CNT and the toxicity assays, and on the oxidative stress.

L’hyperglycémie associée au diabète de type 2 induit des complications vasculaires menant à des désordres cérébrovasculaires, comme l’accident vasculaire cérébral. En effet, l’hyperglycémie altère l’intégrité de la barrière hémato-encéphalique, et les cellules endothéliales cérébrales qui la composent sont particulièrement affectées. Le stress oxydant et l’état pro-inflammatoire engendrés par l’hyperglycémie jouent un rôle causal. Dans ce contexte, un intérêt croissant est accordé aux polyphénols d’origine végétale qui pourraient exercer une action antioxydante et anti-inflammatoire protectrice. L’objectif du travail de thèse était d’évaluer l’impact de l’hyperglycémie sur des marqueurs redox, inflammatoires et vasoactifs des cellules endothéliales cérébrales. L’action protectrice de l’insuline en tant qu’hormone hypoglycémiante clé a été explorée. De plus, nous avons étudié le rôle protecteur de polyphénols antioxydants extraits de la plante médicinale Antirhea borbonica de La Réunion. Pour ce faire, un modèle de cellules endothéliales cérébrales murines ainsi qu’un modèle animal d’ischémie cérébrale en condition d’hyperglycémie ont été utilisés. Nos résultats ont montré que l’hyperglycémie a induit un stress oxydant et une réponse pro-inflammatoire contribuant à une altération de la fonction endothéliale. Plusieurs cibles moléculaires ont été identifiées dont les protéines redox Nox4, Cu/ZnSOD, MnSOD, catalase et HO-1 ainsi que les facteurs vasoactifs ET-1, eNOS et NO. L’implication des médiateurs de signalisation Nrf2, AMPK, PI3K, JNK, ERK, p38 MAPK, NFκB et de la cytokine pro-inflammatoire IL-6 a été mise en évidence. L’insuline et les polyphénols ont exercé des effets antioxydants et anti- inflammatoires protégeant la fonction endothéliale. En situation d’ischémie cérébrale, l’hyperglycémie a exacerbé la dérégulation de l’état redox et pro-inflammatoire cérébral, associée à une altération de la barrière hémato-encéphalique. De plus, l’hyperglycémie a aggravé le déficit neurologique, le volume de l’infarctus cérébral et la transformation hémorragique. Les polyphénols ont exercé un rôle protecteur. L’acide caféique et son métabolite circulant, l’acide férulique, détectés au niveau cérébral, pourraient rendre compte de cette action protectrice. Des travaux complémentaires ont montré que les polyphénols protègent également contre l’altération de la fonction de cellules endothéliales aortiques humaines et la perte de vasorelaxation d’anneaux aortiques isolés de souris exposés à une hyperglycémie associée aux lipopolysaccharides de la bactérie Escherichia coli, qui sont des endotoxines liées de manière causale au contexte diabétique. En conclusion, ce travail de thèse a mis en évidence l’effet délétère de l’hyperglycémie sur la fonction endothéliale et le rôle protecteur de polyphénols antioxydants. L’utilisation des modèles expérimentaux développés permettra d’approfondir l’exploration des voies moléculaires impliquées et d’identifier de possibles cibles thérapeutiques innovantes. / Type 2 diabetes promotes vascular complications, leading to cerebrovascular disorders such as stroke. Indeed, hyperglycemia alters the blood-brain barrier integrity by deregulating the cerebral endothelial cell function. Oxidative stress and pro-inflammatory response may play a causal role. Thus, the biological effect of plant polyphenols known to exert antioxidant and anti-inflammatory capacities is of high interest. We evaluated the impact of hyperglycemia on the production of redox, inflammatory and vasoactive markers of cerebral endothelial cells, and the protective effect of polyphenols from the medicinal plant Antirhea borbonica from Reunion island. The murine bEnd.3 cerebral endothelial cells and an ischemia-reperfusion mouse model exposed to hyperglycemia were used. Our results demonstrated that hyperglycemia induced an oxidative stress and a pro-inflammatory state, leading to cerebral endothelial dysfunction through the activation of specific signaling molecules. Importantly, polyphenols extracted from Antirhea borbonica counteracted hyperglycemia deleterious effects and protected cerebral endothelial cells. Moreover, hyperglycemia exacerbated oxidative stress and pro-inflammatory state promoting cerebrovascular damages and loss of endothelial barrier integrity in ischemia-reperfusion mice model. Polyphenols exerted antioxidant and anti-inflammatory activities, attenuating cerebrovascular damages. These findings suggest that polyphenols extracted from Antirhea borbonica exerted protective effects on cerebral endothelial cells and an ischemia-reperfusion mouse model against deleterious effects of hyperglycemia.

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La sclérose en plaques (SP) est une maladie inflammatoire du système nerveux central (SNC) affectant plus de 2,5 millions de personnes dans le monde. Bien que son étiologie soit inconnue, de nombreuses données supportent la contribution des réponses immunitaires à la maladie. La présence de leucocytes au sein des lésions de démyélinisation est un marqueur neuropathologique de la SP. Les traitements actuels diminuent les exacerbations de la SP mais échouent à arrêter sa progression. Ainsi, il est essentiel d’identifier des nouvelles voies contribuant à la pathologie de la SP. NKG2D est un récepteur co-activateur de cellules effectrices qui participent à la surveillance immunitaire. Cependant, cette voie peut contribuer à l’inflammation et aux lésions tissulaires. Le blocage ou l’élimination du NKG2D réduit la gravité de la maladie dans des modèles animaux de SP. Dans la pathologie humaine, des lymphocytes T CD4+ et CD8+ NKG2D+ sont présents dans les lésions et élevés dans le sang périphérique des patients atteints de la forme cyclique de SP durant une poussée. Au moins un ligand du NKG2D (NKG2DL) est exprimé par des oligodendrocytes dans des lésions et les lymphocytes T CD8+ causent la mort in vitro des oligodendrocytes humains de façon NKG2D dépendante. On ignore encore si d’autres cellules neurales expriment des NKG2DL et sont susceptibles à la reconnaissance par le NKG2D. Dans cette thèse, nous avons investigué la contribution de la voie NKG2D à la pathologie de la SP. La première partie présente la caractérisation de l’expression des NKG2DL au sein du SNC. Nous avons trouvé des niveaux élevés de ULBP4, mais aucun autre ligand, dans les lésions et la matière blanche d’apparence normale chez des patients SP. Nous avons identifié des déclencheurs potentiels de l’expression de ULBP4 par les astrocytes. ULBP4 soluble est détecté dans le liquide céphalo-rachidien, et des essais fonctionnels ont démontré sa capacité à renforcer la sécrétion de cytokines inflammatoires par les lymphocytes T CD8+. Dans la seconde partie, nous avons caractérisé les lymphocytes T exprimant NKG2D dans le sang de patients atteints des formes cycliques et progressives de la SP ainsi que chez des sujets témoins. Bien que nous ayons trouvé des proportions similaires, les lymphocytes T NKG2D+CD4+ de patients SP avaient un phénotype mémoire activé associé aux profils Th1 et Th1/Th17. Les lymphocytes T NKG2D+CD8+ étaient diminués chez les patients atteints de la forme cyclique. Cette population affichait une expression prédominante du granzyme B et manifestait des capacités de dégranulation envers les astrocytes exprimant ULBP4. Finalement, les analyses d’imagerie en temps réel ont révélé un rôle pour NKG2D et son ligand ULBP4 dans les interactions durables entre les astrocytes et les lymphocytes T CD8+ humains. Nos travaux identifient un nouveau mécanisme dans le dialogue entre ces types cellulaires. Globalement, ce projet de thèse présente une caractérisation en profondeur de la voie NKG2D dans la SP. De plus, il fournit de nouvelles preuves quant à l’implication de ULBP4 dans la pathophysiologie de la SP. De nouvelles investigations contribueront à élucider la validité de ULBP4 en tant que cible thérapeutique. / Multiple sclerosis (MS) is a neuroinflammatory disease of the central nervous system (CNS) that affects more than 2.5 million people worldwide. Despite its unknown etiology, numerous evidences point to aberrant immune responses that contribute to the typical tissue damage. Indeed, the presence of infiltrating immune cells within the characteristic focal demyelinating lesions is a pathological hallmark of MS. Current treatments, which target the immune system, generally control disease exacerbations, but have failed to stop progression. Therefore, it is essential to identify common immune pathways that contribute to MS pathology. NKG2D is a co-activating receptor of immune cells that plays a critical role in immune surveillance. Nevertheless, aberrant NKG2D-mediated responses can contribute to inflammation and tissue damage. Various studies have implicated the NKG2D pathway in MS. NKG2D blocking or depletion reduced disease severity in various EAE models, a commonly used animal model of MS. In the human pathology, NKG2D+CD4+ and CD8+ T lymphocytes have been found in MS lesions and are upregulated in the peripheral blood of RRMS patients under relapse. Moreover, at least one NKG2DL has been observed in oligodendrocytes from MS lesions, which were found near CD8+ T lymphocytes. Furthermore, in vitro studies have demonstrated NKG2D-dependent killing of human oligodendrocytes by CD8+ T lymphocytes. Whether other neural cells express NKG2DL and can thus be susceptible to NKG2D-mediated recognition was still unknown. In this thesis, we investigated further the contribution of the NKG2D pathway to the pathobiology of MS. The first part of this project consisted in the evaluation of NKG2DL expression within the CNS. We found upregulated levels of ULBP4, and no other NKG2DL, in MS lesions and normal appearing white matter from MS patients. Moreover, we identified potential triggers observed in MS lesions that could impact on ULBP4 expression. Soluble ULBP4 was also found in the cerebrospinal fluid, and functional assays demonstrated its capacity to boost inflammatory cytokines secretion by CD8+ T lymphocytes. In the second part, we performed a deep characterization of CD4+ and CD8+ T lymphocytes expressing NKG2D in blood samples from relapsing-remitting and progressive forms of MS as well as age and sex matched healthy controls. Despite finding similar proportions, NKG2D+CD4+ T lymphocytes from MS patients exhibited an activated memory phenotype associated with Th1 and Th1/Th17 responses. In contrast, NKG2D+CD8+ T lymphocytes were reduced in RRMS patients. This subset displayed a predominant granzyme B expression irrespective of the donors’ group, and exhibited degranulating capacities toward ULBP4-expressing astrocytes. Finally, live imaging analysis revealed a role for NKG2D and its ligand ULBP4 in the establishment of long-lasting interaction between astrocytes and CD8+ T lymphocytes. This provides a new mechanism involved in the dialogue between these cell types. Overall, this thesis project provides a deep characterization of the NKG2D pathway in relapsing-remitting and progressive MS patients. Moreover, it provides new evidence for the involvement of ULBP4, a specific NKG2DL, in the pathophysiology of MS. Further investigations will contribute to elucidate the validity of ULBP4 as a therapeutic target in MS.