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No description available.

<p>Inflammatory Bowel Disease (IBD) is a chronic intestinal inflammation that has caused many challenges for healthcare providers in treating the disease and also altered the quality of life of the patients. The cure for IBD is still symptomatic-based; the causes mechanism and pathogenesis of IBD are to be further investigated. Currently, IBD has been considered as an excessive immune response to commensal flora that in normal condition is tolerable to the host. Antigen presenting cells (APCs) play an important role in the pathogenesis of IBD. Macrophage is one of the professional APCs that present antigen information to T cells and induce the T cell subtype proliferation. Aside from this role, macrophages also phagocytose pathogens and clean cell debris in thebody.</p> <p>T cell immunoglobulin and mucin domain (TIM)-4 is a glycoprotein expressed on the surface of macrophage, which recognizes phosphatidylserine (PS) that is expressed mainly on the surface of the early apoptotic cell phospholipid membrane; the latter is a negatively charged molecule that can bind to the TIM-4 to enhance the phagocytosing activity. In IBD, the loss of intestinal epithelial cells (IECs) due to apoptosis is prominent in the site of inflammation especially in ulcerative colitis (UC).</p> <p>The aim of this study is to elucidate whether there is an increase of TIM-4 expression in colitis mice model after exposure to excessive number of apoptotic IECs and whether TIM-4 plays a role in the development of colitis in mice.</p> <p>The expression of TIM-4 is measured with several tests; including flow cytometry, immunohistochemistry, western blotting and real time RT-PCR. In the first step, we tried to see if there is a difference in the TIM-4 expression in colitis mice and ethanol control mice. After the association was established, we further observed the role of TIM-4 in the pathogenesis of IBD by injecting TIM-4+ macrophages into the mice prior to inducing a mild colitis in the mice and finally injected neutralizing anti TIM-4 antibodies to block the available TIM-4 receptors.</p> <p>We found that TIM-4 expression was higher in a colitis mouse model compared to the control. Also by injecting TIM-4+ macrophages into the mice, the frequency of intestinal T regulatory (Treg) cells was decreased significantly. Finally in the group treated with anti-TIM-4 neutralizing antibodies prior to colitis induction, the frequency of intestinal Treg cells increased significantly and the inflammation response was less severe than the colitis control group. This study revealed, for the first time in the world, that TIM-4 expression in the colon of colitis mice was significantly increased, which suppressed Tregs and promoted T effector cells.</p> / Master of Science in Medical Sciences (MSMS)

Clostridium perfringens is the most common cause of gas gangrene (clostridial myonecrosis), a disease that begins when ischemic tissues become contaminated with C. perfringens. C. perfringens quickly multiplies in ischemic tissues and spreads to healthy areas, leading to high levels of morbidity and mortality. As a species, the bacterium can synthesize thirteen different toxins. The alpha toxin (PLC) and perfringolysin O (PFO) are thought to be important virulence factors in gangrene. We wished to understand how C. perfringens is capable of avoiding killing by the host immune system, and determine if PLC and PFO play a role in this avoidance. We found C. perfringens was not killed by J774-33 cells or mouse peritoneal macrophages under aerobic or anaerobic conditions. Using electron microscopy, we showed that C. perfringens could escape the phagosome of J774-33 and mouse peritoneal macrophages. We believe the ability of C. perfringens to survive in the presence of macrophages is due to its ability to escape the phagosome. Using a variety of inhibitors of specific receptors, we identified those used by J774-33 cells to phagocytose C. perfringens. The scavenger receptor, mannose receptor(s), and complement receptor (CR3) were involved in the phagocytosis of C. perfringens. To determine if PFO or PLC were involved in the ability of C. perfringens to survive in the presence of macrophages, we constructed C. perfringens strains lacking these toxins. The ability of C. perfringens to survive in the presence of J774-33 cells is dependent on PFO, while survival in mouse peritoneal macrophages is dependent on PFO and PLC. The ability of C. perfringens to escape the phagosome of J774-33 cells and mouse peritoneal macrophages is mediated by either PFO or PLC. Using a mouse model, we found that PFO and PLC were necessary for C. perfringens to survive in vivo using infectious doses 1000 times lower than those required to initiate a gangrene infection. We propose that PFO and PLC play a critical role in the survival of C. perfringens during the early stages of gangrene infections, when phagocytic cells are present and bacterial numbers are low. / Ph. D.

Thèse numérisée par la Direction des bibliothèques de l'Université de Montréal.

The quality of marine bathing water is currently assessed by monitoring the levels of faecal indicator bacteria. Among other drawbacks, results are retrospective using the traditional culture based methods. A rapid method is thus needed as an early warning to bathers for bacterial contamination in marine bathing waters. Total lipopolysaccharide (LPS) was chosen here as a potential general biomarker for bacterial contamination. Levels of total LPS, measured using a Kinetic QCL™ Limulus Amebocyte Lysate (LAL) assay, highly correlated with enumerated Escherichia coli and Bacteroides species. Levels of LPS in excess of 50 EU mL-1 were found to equate with water that was unsuitable for bathing under the current European Union regulations. Results showed that monitoring the levels of total LPS has a potential applicability as a rapid method for screening the quality of marine bathing water. More importantly, the LAL assay overcome the retrospective results when using culture based assessment since the LAL assay takes less than 30 minutes. Although false positive events were not detected, the occurrence of a false positive has been hypothesised, hence a more specific faecal biomarker was also investigated. LPS of five Bacteroides species (B. fragilis, B. caccae, B. ovatus, B. xylanisolvens and B. finegoldii) isolated from marine bathing waters samples were successfully profiled and showed high similarity between isolates in LPS gel electrophoresis banding pattern. Similar results were shown when investigating the endotoxic activity of Bacteroides species with the Kinetic QCL™ LAL assay. The potential biological relevance of Bacteroides LPS was also investigated in cell culture models indicating that Bacteroides showed similar induction of proinflammatory cytokines (TNF-α, IL-6 and IL-1α) and generally the biological activity was approximately 100 fold less than E. coli LPS. In addition, an ELISA assay was designed for the detection of Bacteroides LPS. Results showed that the Bacteroides LPS has a high potential to be used as a faecal biomarker, however, further work is required to develop a fully functional assay. The potential biological relevance of LPS present in contaminated bathing waters was also investigated in cell culture models. Results showed that there is a significant difference in the production of proinflammatory cytokines in comparison to “clean” bathing waters. Thus, results suggest that the European Directive regulations should be extended to cover the levels of total LPS in bathing waters to assure safety to the users of marine recreational water.

Le fer est un oligo-élément nécessaire pour le fonctionnement normal de toutes les cellules de l'organisme et joue un rôle essentiel dans de nombreuses fonctions biologiques. Cependant, le niveau de fer dans le corps doit être bien réglé, sinon la carence en fer entraine des divers états pathologiques tels que l'anémie et la diminution de l’immunité. D'autre part, une surcharge en fer potentialise la multiplication des germes, aggrave l’infection et la formation de radicaux libres ayant des effets toxiques sur les cellules et leurs composants, ce qui favorise les maladies cardio-vasculaires, l'inflammation et le cancer. L'hepcidine (HAMP), un régulateur négatif de l'absorption du fer, induit la dégradation de la ferroportine (FPN), le seul exportateur connu de fer ce qui réduit sa libération par les macrophages et inhibe son absorption gastro-intestinale. HAMP est synthétisé principalement par les hépatocytes, mais aussi par les macrophages. Cependant, il y a très peu de données sur la façon dont HAMP est régulé au niveau des macrophages. Plus récemment, nous avons constaté que l’induction de l’hepcidin dans le foie par le polysaccharide (LPS) est dépendante de la voie de signalisation médiée par « Toll-like receptor 4 » (TLR4). Grâce au TLR4, le LPS induit l'activation des macrophages qui sécrètent de nombreuses différentes cytokines inflammatoires, y compris Interleukine 6 (IL-6), responsable de l'expression de HAMP hépatique. Dans le premier chapitre de la présente étude, nous avons étudié la régulation de HAMP dans la lignée cellulaire macrophagique RAW264.7 et dans les macrophages péritonéaux murins stimulés par différents ligands des TLRs. Nous avons constaté que TLR2 et TLR4 par l'intermédiaire de la protéine adaptatrice « myeloid differentiation primary response gene 88 » (MyD88) activent l'expression de HAMP dans les cellules RAW264.7 et les macrophages péritonéaux sauvages murins, tandis que cette expression a été supprimée dans les macrophages isolés des souris TLR2-/-, TLR4-déficiente ou MyD88-/-. En outre, nous avons constaté que la production d'IL-6 par les cellules RAW264.7 stimulées avec du LPS a été renforcée par l’ajout des quantités élevées de fer dans le milieu de culture. Au cours de l’inflammation, le niveau de HAMP est fortement augmenté. Ainsi, lorsque l'inflammation persiste, l’expression de HAMP continue à être activée par des cytokines pro-inflammatoires conduisant à une hyposidérémie. Malgré que cette dernière soit considérée comme une défense de l'hôte pour priver les micro-organismes de fer, celle ci cause un développement d'anémies nommées anémies des maladies chroniques. Ainsi, dans le deuxième chapitre de la présente étude, nous avons étudié l'implication des TLRs et leurs protéines adaptatrices MyD88 et TIR-domain-containing adapter-inducing interferon-β (TRIF) dans le développement des hyposidérémies. En utilisant des souris déficientes en MyD88 et TRIF, nous avons montré que les voies de signalisations MyD88 et TRIF sont essentielles pour l’induction de HAMP par le LPS. Malgré l'absence de HAMP, les souris déficientes ont été capables de développer une hyposidérémie, mais la réponse des souris déficientes en MyD88 a été très légère, ce qui indique l'exigence de cette protéine pour assurer une réponse maximale au LPS. En outre, nous avons constaté que la signalisation MyD88 est nécessaire pour le stockage du fer au niveau de la rate, ainsi que l'induction de lipocaline 2 (LCN2), qui est une protéine impliquée dans la fixation du fer pour limiter la croissance bactérienne. Indépendamment de MyD88 ou TRIF, l'activation de TLR4 et TLR3 a conduit, au niveau de la rate, à une diminution rapide de l’expression de FPN et du « Human hemochromatosis protein » (HFE) qui est une protéine qui limite la séquestration du fer cellulaire à partir de la circulation. Cependant, malgré cette baisse d’expression, le manque de la signalisation MyD88 a altéré de manière significative la réponse hyposidérémique. En établissant le rôle des TLRs et de la protéine adaptatrice MyD88 dans la diminution du taux du fer sérique au cours de la réponse inflammatoire, nous avons remarqué qu’en réponse au surcharge en fer les souris déficientes en MyD88 accumulent de manière significative plus de fer hépatique par rapport aux souris sauvages, et cela indépendamment des TLRs. Ainsi, dans le troisième chapitre de la présente étude, nous avons étudié le phénotype observé chez les souris déficientes en MyD88. Nous avons trouvé que l'expression de HAMP chez ces souris a été plus faible que celle des souris de type sauvage. Pour cela, nous avons exploré la signalisation à travers la voie du « Bone Morphogenetic Proteins 6 » (BMP6) qui est considérée comme étant la voie fondamentale de la régulation de HAMP en réponse aux concentrations du fer intracellulaires et extracellulaires et nous avons trouvé que l'expression protéique de Smad4, un régulateur positif de l'expression de HAMP, est significativement plus faible chez les souris MyD88-/- par rapport aux souris sauvages. En outre, on a montré que MyD88 interagit avec « mothers against decapentaplegic, Drosophila, homolog 4 » (Smad4) et que cette interaction est essentielle pour l’induction de HAMP à travers la voie BMP6. En conclusion, notre étude montre que l'expression de HAMP dans les macrophages est régulée principalement par TLR2 et TLR4 à travers la voie MyD88 et que l'accumulation du fer dans les macrophages peut affecter les niveaux des cytokines pro-inflammatoires. En outre, nos analyses démontrent que le développement d’hyposidérémie en réponse au LPS se produit par l'intermédiaire d’un mécanisme dépendant de MyD88 qui est dissociée de la production de cytokines et de HAMP. En plus, nos recherches montrent que MyD88 est nécessaire pour l'expression de Smad4 et cela pour garantir une réponse optimale à travers la signalisation BMP6, conduisant ainsi à une expression adéquate de HAMP. Enfin, la protéine MyD88 joue un rôle crucial dans, la régulation de HAMP au niveau des macrophages, la diminution du taux du fer sérique en réponse au LPS et le maintien de l'homéostasie du fer. / Iron is an oligoelement necessary for normal functioning of all body cells and plays an essential role in many biological functions. However, the level of iron in the body must be well regulated, otherwise iron deficiency results in various pathological conditions such as anemia and decreased immunity. On the other hand, iron overload potentiates the multiplication of germs and infection worsens, and the formation of free radicals with toxic effects on cells and their components, thus promoting cardiovascular diseases, inflammation and cancer. Hepcidin (HAMP), a negative regulator of iron absorption, induces the degradation of the only known iron exporter ferroportin (FPN) resulting in the reduction of iron release by macrophages and in the inhibition of its gastrointestinal uptake. HAMP is synthesized mainly by hepatocytes but also by macrophages. However, there are very little data about how HAMP is regulated in macrophages. More recently, we found that HAMP induction in the liver by polysaccharide (LPS) is dependent on the signaling pathway mediated by Toll-like receptor 4 (TLR4). Through TLR4, LPS induces the activation of macrophages which will secrete many different inflammatory cytokines, including Interleukine 6 (IL-6), responsible of hepatic HAMP expression. In the first chapter of the present study, we investigated HAMP regulation in the RAW264.7 macrophage cell line and in murine peritoneal macrophages stimulated with different TLR ligands. We found that TLR2 and TLR4 signaling through the myeloid differentiation primary response gene 88 (MyD88) adaptor protein activate hepcidin expression in RAW264.7 cells and in wild-type murine peritoneal macrophages, while this expression was abolished in TLR2−/−, TLR4-deficient or MyD88−/− isolated macrophages. Moreover, we found that IL-6 production by RAW264.7 cells stimulated with LPS was enhanced by high amounts of iron present in the culture medium. During inflammation, the level of HAMP is greatly increased. Thus, when inflammation persists, HAMP expression continues to be activated by proinflammatory cytokines leading to hypoferremia. Despite that the latter is considered as host defence to deprive microorganisms of iron, this will cause the development of anemia of chronic disease. Thus, in the second chapter of the present study, we investigated the involvement of TLRs signaling through their adaptor proteins MyD88 and TIR-domain-containing adapter-inducing interferon-β (TRIF) in the development of hypoferremia. Using MyD88-deficient and TRIF-deficient mice, we show that MyD88 and TRIF signaling pathways are critical for HAMP up-regulation by LPS. Despite the lack of HAMP, both deficient mice were able to develop hypoferremia; however the response in MyD88 deficient mice was very mild, indicating the requirement of MyD88 adaptor protein for the acute hypoferremic response to LPS. Furthermore, we found that MyD88 signaling is required for iron sequestration in the spleen and the induction of lipocalin 2 (LCN2) which is a protein involved in iron sequestration that in turn limits bacterial growth. Independently of MyD88 or TRIF, the activation of TLR4 and TLR3 signaling resulted in rapid down-regulation of splenic FPN and the Human hemochromatosis protein (HFE) which is a protein that limit cellular iron uptake from the circulation. However, despite the latter down-regulation, the lack of MyD88 signaling significantly impaired the hypoferremic response. While establishing the role of TLRs signaling through MyD88 adaptor protein in the acute phase of hypoferemia, we noticed that MyD88-deficient mice accumulate significantly more iron in their livers than wild-type mice in response to iron loading, and this independently of TLRs. Thus, in this third chapter of the present study, we studied the phenotype observed in MyD88-deficient mice. We found that HAMP expression in MyD88-deficient mice was lower than wild-type mice. Regarding this result, we explored the Bone Morphogenetic Proteins 6 (BMP6) signaling which is considered to be the fundamental pathway regulating HAMP levels in response to intracellular and extracellular iron concentrations and we found by western blot that Smad4 expression is significantly lower in MyD88-/- mice when compared to wild-type mice. We further show that MyD88 interacts with the mothers against decapentaplegic, Drosophila, homolog 4 (Smad4), a positive regulator of HAMP expression, and that this interaction is critical for HAMP induction through the Smad4 iron-sensing pathway. In conclusion, our study shows that HAMP expression in macrophages is regulated mainly through TLR2 and TLR4 receptors via the MyD88-dependent signaling pathway and that autocrine regulation of iron accumulation in macrophages by HAMP may affect the levels of proinflammatory cytokine production. Furthermore, our analysis shows that the development of hypoferremia during LPS response occur via a MyD88-dependent mechanism that is dissociated from peripheral cytokine production and hepatic HAMP induction. This work shows that MyD88 is required for Smad4 expression to guarantee an optimum response to BMP6 signaling, leading to adequate HAMP expression. Finally, the MyD88 adopter protein plays a crucial role in the regulation of HAMP expression by macrophages, the development of the hypoferremic response by LPS and the maintenance of iron homeostasis.

La candidose oro-pharyngée (COP) est l’infection opportuniste la plus répandue chez les patients infectés au VIH-1. Un modèle de COP chez la souris transgénique (Tg) exprimant une partie du génome du VIH-1 (CD4C/HIVMutA) est maintenant disponible. Grâce à ce modèle, il est possible d’étudier les perturbations quantitatives et fonctionnelles des macrophages exprimant les gènes nef, rev et env du VIH-1 dans le contexte d’une COP. Cette étude démontre que la présence du transgène n’influence pas le pourcentage des macrophages dans la muqueuse buccale et le petit intestin, malgré le fait que la charge buccale de C. albicans soit significativement plus élevée chez les souris Tg. Cependant, l’expression du transgène cause une diminution de la production de H2O2 par les macrophages, ainsi que l’augmentation de la production de la cytokine proinflammatoire IL-6 et de la chimiokine MCP-1. / Oro-pharyngeal candidiasis (OPC) is the most common opportunistic infection in HIV-1 infected patients. An OPC model using transgenic mice (CD4C/HIVMutA) expressing selected genes of the HIV-1 genome is now available. Using this model, it is now possible to study potential quantitative and functional disturbances in macrophages expressing the nef, rev and env genes of HIV-1 in the context of OPC. This study shows that transgene expression does not affect quantitative percentage values of macrophages in the oral mucosa and the small intestine, although burdens of C. albicans loads are increased in Tg mice. Transgene expression does induce diminished H2O2 production in macrophages, while increasing production of the proinflammatory cytokine IL-6 and the chemokine MCP-1.

Coronary artery disease (CAD) is caused by atherosclerosis, a chronic inflammatory response to modified lipoproteins. A key pathophysiological event is the lipid-induced transformation of macrophages into lipid-laden foam cells and their accumulation in atherosclerotic plaques. Heritable CAD risk is associated with common genetic variants at over 40 genomic loci; the underlying causal mechanisms remain largely unknown and could affect transcriptional regulation in foam cells. Epigenetic and gene expression changes were measured in primary human macrophages before and after exposure to atherogenic, oxidized low-density lipoprotein—with resultant foam cell formation. This unbiased approach involved open chromatin mapping with formaldehyde-assisted isolation of regulatory elements with enhancer and transcription factor mapping using chromatin immuno-precipitation. Foam cell formation was associated with changes in a subset of open chromatin and enhancer sites that were strongly correlated with expression of nearby genes. OxLDL-regulated enhancers were enriched for several transcription factors—including C/EBP-beta— that have no previously documented role in foam cell formation. OxLDL exposure up-regulated C/EBP-beta expression and increased C/EBP-beta binding across the genome, most prominently around genes involved in inflammatory response pathways. Variants at CAD-associated loci were enriched in the subset of oxLDLregulated open chromatin sites. These included rs72664324 in an oxLDL-induced super-enhancer at the PPAP2B locus. OxLDL increased C/EBP-beta binding at rs72664324. C/EBP-beta binding, enhancer activity and oxLDL-induced upregulation of PPAP2B were stronger with the protective A allele of rs72664324. The PPAP2B protein product LPP3 was expressed in foam cells in human atherosclerotic plaques and was upregulated by oxLDL exposure in macrophages, so increasing the degradation of pro-inflammatory mediators. I also found several other CAD risk candidate genes were regulated by oxLDL: Phosphatase and actin regulator 1 (PHACTR1) and macrophage inducible Ca²⁺ dependent C-type lectin (Mincle). This led us to find a novel expression-quantitative-trait locus for PHACTR1 in macrophages and define new glycolipid ligands for Mincle. Our results demonstrate a genetic mechanism contributing to CAD risk at the PPAP2B locus and highlight the value of integrating gene expression and epigenetic changes to study disease processes involving pathogenic environmental stimuli.