La consommation excessive d’alcool concerne environ 20% de la population adulte en France. Il est établi qu’une consommation excessive aigue d’alcool engendre une augmentation de la morbi-mortalité en cas d’infection ou de traumatisme. La consommation chronique d’alcool augmente le risque de cancers et d’infection.Toutes ces conséquences médicales sont intimement liées à une altération des défenses de l’hôte induite par l’alcool. L’ingestion d’alcool et ses métabolites engendre une modification de la flore digestive appelée dysbiose ainsi qu’une augmentation de la perméabilité digestive. De cet effet local découle une augmentation du passage d’endotoxines dans le système veineux portal et systémique. Ainsi l’ensemble des éléments impliqués dans la réponse immunologique sont impactés de façon locale (Foie) et systémique par cette endotoxinémie chronique. Deux axes de recherche ont été privilégiés dans cette thèse: La translocation bactérienne et les sous-populations monocytaires. L’originalité de ce travail est l’étude de la cinétique des modifications immunologiques, chez des sujets alcoolo-dépendants (AD) hospitalisés pour un sevrage en alcool durant 2 à 6 semaines.La translocation bactérienne (TB) a été étudiée par l’analyse itérative de marqueurs sériques témoins de celle-ci: l’ADN 16S ribosomial en PCR temps réel, les taux sériques de LBP (LPS-Binding-Protein) et de CD14 soluble par ELISA. Les prélèvements veineux ont été effectués à jeun chez des sujets AD à J0 de leur sevrage et après 4 puis 6 semaines de sevrage. Avant le sevrage (J0) les 3 marqueurs sont plus élevés que dans une population témoin (p<0.001). Après 6 semaines de sevrage, les taux de LBP (p=0.04) et sCD14 (p=0.001) diminuent de manière significative sans revenir aux taux de la population témoin. La consommation de cannabis dans le mois précédent le sevrage est associée à une plus grande diminution des marqueurs LBP et sCD14 au cours du sevrage en alcool.Notre étude confirme une majoration de la TB chez les sujets AD non sevrés et l’impact potentiel du cannabis sur celle-ci. Elle montre également que le délai de sevrage de 6 semaines ne permet pas un retour à la normale des marqueurs de la TB.La répartition, le phénotype et la fonctionnalité des sous-populations monocytaires sanguines ont été étudiés à J0 et après 14 jours de sevrage en alcool chez des sujets AD. Avant le sevrage (J0), la fréquence des monocytes classiques (CD14+CD16-) est diminuée alors que celle des non classiques (CD14dimCD16+) est augmentée chez les sujets AD comparativement aux sujets contrôles. La fréquence des monocytes exprimant les TLR-2 et -4 est réduite chez les sujets AD. La sécrétion basale des cytokines IL-1, IL-6 et TNF est comparable chez les sujets AD et contrôles. En revanche après stimulation in vitro des monocytes par les ligands des TLR-2 et 4, respectivement peptidoglycanes (PGN) et lipopolysaccharides (LPS), la sécrétion d’IL-6 et de TNF est augmentée chez les sujets AD. Le sevrage de 14 jours restaure partiellement la distribution des sous-populations monocytaires. Nos résultats indiquent que la consommation chronique d’alcool altère la distribution, le phénotype et la fonctionnalité des monocytes chez les sujets AD, ces altérations s’améliorent en 14 jours de sevrage mais ne reviennent pas à la normale.Mon travail de thèse confirme l’impact de la consommation chronique d’alcool sur la translocation bactérienne et sur la réponse immune chez l’homme. Il précise la nature et la cinétique de cet impact. Mes résultats suggèrent également que le délai de retour à la normale semble être supérieur à 6 semaines. De nouvelles études permettront de mettre en évidence une cinétique plus précise de ces améliorations biologiques. Les résultats permettent d’envisager des recommandations cliniques quant à une durée d’abstinence pré-opératoire en cas de chirurgie programmée par exemple. / Excessive alcohol consumption concerns about 20% of the French adult population. An acute excessive alcohol consumption named “binge drinking” causes an increase in mortality and morbidity in case of trauma or infection. Chronic alcohol consumption causes an increased risk and severity of infections and increased risk of cancer. All these medical consequences are linked to changes in host defense induced by alcohol consumption. Indeed, alcohol and its metabolites generate modifications of the gut microbiota and increased gut permeability. This local effect causes an increase in endotoxin translocation into portal and systemic blood. Thus, all elements involved in the immune response, and in particular the monocytes as cellular component acting as first line of defense of the immune system, are affected by this chronic endotoxemia. Two research axes were studied during my thesis: Microbial translocation and monocyte subsets. The originality of this work was to study the kinetic of immunological changes induced by alcohol withdrawal in alcohol-dependent (AD) subjects hospitalized for an alcohol withdrawal during 2-6 weeks.Microbial translocation (MT) was studied by iterative analysis of serum markers: Bacterial 16S rDNA levels were measured using qPCR, Lipopolysaccharides-Binding protein (LBP) and soluble CD14 were quantified using ELISA. Blood samples were collected in fasten AD subjects at D0 of alcohol withdrawal and after 4 and 6 weeks of alcohol withdrawal. At D0, the 3 markers of MT were higher in AD subjects than in healthy controls (P<0.001). After 6 weeks of alcohol withdrawal, the serum level of LBP (p=0.04) and sCD14 (p=0.001) were significantly decreased but did not reach rates of healthy controls (HC). Cannabis use during the last month before alcohol withdrawal was associated with a greater decrease in sCD14 and LBP upon alcohol withdrawal. My study confirms an increase in MT in AD subjects and the potential impact of cannabis on this phenomenon, and shows that a 6-weeks abstinence period is not sufficient to return to normal blood biological parameters.Whether and how blood monocyte subsets were impaired in AD patients were studied as well as their evolution after alcohol withdrawal. The CD14+CD16- subset was decreased whereas the CD14dimCD16+ subset was expanded (p<0,001) in AD compared to HC. The frequencies of TLR2- and TLR4-expressing monocytes were reduced in AD compared to HC. Although the basal production of IL-1, IL-6 and TNF by monocytes in AD was comparable to HC, the PGN- and LPS-mediated IL-6 and TNF production were increased in AD. Frequencies of IL-6-expressing monocytes were higher in AD than HC. Alcohol withdrawal partially restored the distribution of monocyte subsets and the frequency of IL-6-producing monocytes, and increased the frequency of TNF-producing cells in response to LPS and PGN stimulation to levels comparable to those in HC. Our findings indicate that chronic alcohol use alters the distribution as well as the phenotypic and functional characteristics of blood monocyte subsets, which are partially restored following 2 weeks of alcohol withdrawal. These studies confirm and specify the impact of chronic alcohol consumption on microbial translocation and on the immune response. Our results also suggest that a period superior to 6 weeks is necessary to reach normal biological parameters. News studies will be necessary to specify the exact kinetic of these biological improvements.These results allow to consider clinical recommendations for a period of abstinence before programmed surgery for example.

Contexte: La leucémie lymphoïde chronique (LLC) est une hémopathie maligne associée à des anomalies dans les réponses immunitaires. Ce qui contribue à la progression de la maladie. Certains LB peuvent induire une inhibition de la réponse anti-tumorale des LT et ainsi promouvoir la progression des tumeurs. Ces LB appelés LB régulateurs partagent des caractéristiques communes avec les LB de LLC.Objectif: L'objectif principal de ce projet est d'évaluer la fonction régulatrice des LB de LLC, afin d’estimer leur influence sur l'absence de réponses anti-tumorales par les cellules T.Méthode: Des modèles de co-culture in vitro ont été développés pour évaluer la capacité desLB à inhiber la prolifération des LT.Résultats: Nos résultats montrent qu’en absence de stimulation, les LB de LLC sont incapables d’inhiber la prolifération des LT contrairement aux LB de témoins. Cependant, deux groupes de patients ont été identifiés après stimulation du TLR-9 des LB. Dans le premier groupe, les LB présentent des fonctions régulatrices défectueuses par rapport aux LB de témoins. Dans le second groupe, aucune activité inhibitrice n’est détectée. L’expression différentielle des gènes associés à la voie TLR-9 entre les deux groupes de patients semble pouvoir expliquer cette dichotomie de réponse. Une corrélation a par ailleurs été retrouvée entre le doublement lymphocytaire et la faible activité régulatrice des LB de LLC.Conclusion: Pour aller plus loin, il convient d'identifier les mécanismes moléculaires endommageant la voie TLR-9. Ces nouvelles données aideront à la compréhension de l’absence de réponse anti-tumorale et des dysfonctionnements immunitaires retrouvés chez les patients. / Background: Chronic lymphocytic leukemia (CLL) is characterized by expansion of CD5+B cells associated with disruption of immune responses, contributing to the immunodeficiency and the disease progression. Regulatory B (Breg) cells may control the anti-tumor responses favoring tumor escape. Intriguingly, CLL B cells share phenotypical characteristics with these cells.Aims: The main focus of this project is to evaluate the regulatory function of CLL B cells, aiming to estimate their influence on the lack of anti-tumor responses mediated by T cells.Methods: In vitro models of co-cultures between T and B cells are used to appraise the regulatory capacity of CLL B cells on T cell proliferation.Results: We determined a defective spontaneous regulatory function for CLL B cells. Two groups of patients have been identified following CpG-ODN stimulation. The first group presents defective regulatory B cell functions compared with control B cells. In the second group, no inhibitory activity is detected. TLR-9 gene expression analysis highlighted differential gene expression between controls and the two groups of CLL patients. Moreover, ours observations indicate that patients with low Breg activity have more aggressive disease.Conclusion: These results suggest alteration of the TLR-9 pathway in CLL B cells. To go further, it will be of interest to identify the molecular mechanisms damaging the TLR-9 pathway. These results would contribute to clarify the lack of anti-tumor immune response found in the CLL patients.

Cationic steroid antimicrobial agents (CSAs) are a family of bile acid derivatives. These compounds are amphiphilic and mimic endogenous antimicrobial peptides. The antimicrobial activities of CSA-13 have been investigated and due to portent bactericidal activities and low toxicity, a large amount of CSA-13 is demanded for clinic trails and other antimicrobial applications. During our studies, we optimized the synthetic route of CSA-13, so that it can be prepared at the kilogram, even in tons scale. We investigated three routes and one of them is suitable for industry, because only recrystallization is needed in the synthesis. Natural killer T cells (NKT cells) are a kind of lymphocyte that bridge the adaptive immune system with the innate immune system. Once stimulated by glycolipids, NKT cells influence immune responses. To search for better glycolipid ligands, scientists have isolated many natural products to get inspiration. Thrautochysides A-C was isolated from a group of marine protists. These compounds have an interesting structure on their sphingosine lipid chains. We finished the iii synthesis of thraustochyside B, and made substantial progress toward the synthesis of thraustochyside A. Toll like receptors (TLRs) are integral components of the innate immune system. They recognize antigens and induce dendritic cells to give immune responses. TLR1, TLR2 and TLR6 recognize lipopetides, and these TLRs function as heterodimers. TLR1/TLR2 dimer recognition gives inflammatory responses, and TLR2/TLR6 dimer recognition gives immunomodulatory responses. We used modeling of TLRs to find a compound, which can fill the lipid binding pockets of the TLR2 and TLR6 dimer. In our study, we found the peptide chain of the antigen Pam2CSK4 can be replaced by a water soluble polyamine, which confirmed the function of the peptide to increase the water solubility.

Tick saliva contains a lot of molecules with antihemostatic and immunosupressive effects.The goal of this thesis is to test the effects of tick salivary cystatins from I.ricinus and I.scapularis on TLR - induced maturation of bone-marrow derived dendritic cells and production of chosen cytokines. Over all, the supressive effect of tick cystatins was observed in relation to TLR-induced maturation of DC. In addition, cystatins enhanced production of IL-10 and attenuated induction of IL-12 cytokines.

Cytomegaloviruses (CMV), the prototypical β-herpesviruses, have co-evolved with their hosts and thus acquired multiple strategies for modulation of the immune response. Viral engagement of pattern recognition receptors (PRR), such as toll-like receptors (TLRs) and cytosolic nucleic acids sensors, initiates the host immune response through activation of elaborate signalling programs. The ensuing inflammatory response is further sustained and amplified through cytokines, such as IL-1β, activating signalling pathways greatly overlapping those utilized by TLRs. The central hypothesis of this thesis is that a viral counter-measure by murine CMV (MCMV) involves specific targeting of TLR- and IL-1β-induced signalling along the MyD88 to NF-κB pathway. To test this hypothesis MCMV inhibition of IL-1β signalling was initially investigated in a fibroblast cell line. It was demonstrated that in MCMV infected cells IL-1β-induced IκBα degradation is largely inhibited. Comparison of productive and non-productive infection showed this modulation requires de-novo viral gene expression beyond the immediate early region. Further investigations utilising a ORF M45 deletion mutant identified viral gene M45 as necessary for mediating the observed modulation of IL-1β- induced IκBα degradation. To further test the hypothesis, studies were extended to include TLR stimulation in the context of bone marrow-derived macrophages (BMDM) infection. It was found that TLR7/9-induced NF-κB activation is inhibited in MCMV infected BMDM. Overall, data presented in this study demonstrate a previously unrecognised MCMV inhibition of IL-1β- and TLR7/9-induced NF-κB activation, and indicate a role for viral gene M45 in mediating this effect.

Les cellules dendritiques plasmacytoïdes (pDCs) font le lien entre l'immunité innée et l'immunité adaptative en produisant de l'interféron de type 1 en grandes quantités ainsi qu'en induisant l'activation et la prolifération des cellules T naïves de manière antigène-spécifique. Les pDCs expriment à haut niveau les récepteurs TLR7 et TLR9 et détectent ainsi les acides nucléiques d'origine virale. Les TLRs 7 et 9, localisés dans le réticulum endoplasmique (RE) à l'état basal, sont relocalisés vers les endosomes tardifs, lors de l'activation, pour initier la signalisation. Ce processus est dépendant de la protéine chaperon UNC93B1. Au cours de ma thèse, j'ai caractérisé une nouvelle molécule de la famille des protéines membranaires associées aux lysomes (LAMP), nommée BAD-LAMP. Cette protéine est, au sein du système immunitaire humain, exprimée spécifiquement dans les pDCs. Contrairement aux autres membres de la famille, BAD-LAMP n'est pas détectée dans les lysosomes mais est retenue dans le RE. J'ai également démontré que BAD-LAMP est régulée négativement lors de l'activation des pDCs induite par un ligand de TLR9. L'utilisation d'un système de cellules HeLa tranfectées et de différents mutants de BAD-LAMP avec des défauts de localisation m'ont permis d'établir que BAD-LAMP et UNC93B1 sont capables d'influencer mutuellement les adressage, par un mécanisme restant à identifier. BAD-LAMP pourrait aussi remplir un rôle de chaperon du complexe UNC93B1-TLR9 et moduler la réponse TLR9. L'étude d'un tel mécanisme de contrôle permettrait de mieux comprendre la régulation fine de la réponse immunitaire. / Plasmacytoid dendritic cells (pDCs) link innate and adaptative immunity by producing large amounts of type-1 interferon and inducing naive T cell activation and proliferation in an antigen-specific manner. pDCs express high levels of TLR7 and TLR9 and thereby sense viral nucleic acids. TLRs 7 and 9, which rest in the endoplamic reticulum (ER) at steady-state, are re-localized to the late endosomal compartment upon activation for signaling. this process is dependent of the interaction between TLRs and the chaperone UNC93B1. During my thesis, I characterized a new molecule of the lysosome-associated membrane protein (LAMP) family, named BAD-LAMP. In the human immune system, this protein is exclusively expressed in pDCs. BAD-LAMP is not detected in lysosomes, as opposed to the other LAMP family members, but is retained in the ER compartment. I also demonstrated that BAD-LAMP is down-regulated after pDCs activation by a TLR9 ligand. Using trnasfered HeLa cells and several mutant forms of BAD-LAMP with localization defects, I etablished that BAD-LAMP and UNC93B1 can influence reciprocally their intercellular trafficking by a yet uncharacterize mechanism. BAD-LAMP could therefore act as a chaperone of UNC93B1-TLR9 complex and moduate the TLR9 response. The study of such a regulatory mechanism could help to understand better the fine tuning of the immune response.

Thèse numérisée par la Division de la gestion de documents et des archives de l'Université de Montréal.

Mémoire numérisé par la Direction des bibliothèques de l'Université de Montréal.

Mémoire numérisé par la Direction des bibliothèques de l'Université de Montréal.

In organisms from insects to vertebrates, Toll-like receptors (TLRs) are primary pathogen detectors that activate downstream pathways, specifically those that direct expression of innate immune effector genes. TLRs also have roles in development in many species. The sea anemone Nematostella vectensis is a useful cnidarian model to study the origins of TLR signaling because its genome encodes a single TLR and homologs of many downstream signaling components, including the NF-κB pathway. In this dissertation, the single N. vectensis TLR (Nv-TLR) is characterized. It is demonstrated that Nv-TLR can activate canonical NF-κB signaling in human cells, the intracellular TIR domain of Nv-TLR can interact with human TLR adapter proteins MAL and MYD88, and the TIR domain of Nv-TLR is required for NF-κB activation. It is shown that the coral pathogen Vibrio coralliilyticus causes a rapidly lethal disease in N. vectensis and that heat-inactivated V. coralliilyticus and bacterial flagellin can activate a reconstituted Nv-TLR-to-NF-κB pathway in human cells. By immunostaining of anemones, Nv-TLR is shown to be expressed in a subset of cnidocytes and many of these Nv-TLR-positive cells also express Nv-NF-κB. Additionally, the nematosome, which is a Nematostella-specific multicellular structure, expresses Nv-TLR, many innate immune pathway homologs, and can engulf V. coralliilyticus. Morpholino knockdown indicates that Nv-TLR also has an essential role during early embryonic development. The characterization of this primitive TLR and identification of a bacterial pathogen for N. vectensis reveal ancient TLR functions and provide a novel model for studying the molecular basis of cnidarian disease and immunity.