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Glycopeptidolipid (GPL) antigens isolated from Mycobacterium intracellulare were used to sensitize BALB/c mice. Primary footpad challenge of mice sensitized with GPL antigens suspended in phosphate buffered saline demonstrated an increased footpad swelling reaction.

Macrophages (Mφ) are a major immune cell involved in anti-tumor responses. Mφ activities such as tumor cytotoxicity. presentation of tumor-associated antigens, and stimulation of anti-tumor lymphocytes are all involved in the battle against tumor growth. However, other Mφ activities such as cell growth promotion, angiogenesis, and suppression of anti-tumor lymphocytes aid in tumor growth. This dissertation discusses how tumors control Mφ activities to create favorable environments for tumor growth. Assessment of tumor- and Mφ-derived molecules has enabled me to design models of communication between tumors, Mφ, and other immune cells. A major research focus was to determine how tumor-derived molecules induce Mφ suppressor activity and control Mφ cytotoxicity. Tumor growth induced Mφ to suppress T lymphocyte proliferation by increasing Mφ production of the suppressor molecules prostaglandin E₂ (PGE₂), nitric oxide (NO), and tumor necrosis factor-α (TNF-α). A major finding was that TNF-α's normal up-regulatory action on T-cell proliferation switched to a suppressor action when Mφ were present. The autocrine action of increased TNF-α levels during tumor growth stimulated Mφ PGE₂ and NO synthesis, which suppressed T-cell proliferation. / Ph. D.

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Glioblastoma is the most common and aggressive type of adult brain tumour. It is currently incurable and requires more effective treatments. Tissue transglutaminase 2 (TGM2) has previously been suggested to have a role in glioblastoma. Previous studies focused on TGM2 expression and inhibition in glioblastoma cells. Here we were interested in TGM2 expression in glioblastoma-associated microglia/macrophages and in identifying the role it plays in the tumor microenvironment. Based on data from bioinformatics, cell culture experiments, immunohistochemistry and immunofluorescence on mouse samples and human samples, we have shown that glioblastoma-associated microglia/macrophages are the major source of TGM2 in the tumor microenvironment. We also identified a novel role for TGM2 in efferocytosis in glioblastoma; this suggests a role for TGM2 in the maintenance of an immunosuppressive environment in this cancer. With this, we hope that further studies will be designed to evaluate the use of TGM2 antagonists as therapeutic agents for glioblastoma.

Le virus Zika est un pathogène qui a récemment retenu l’attention de la communauté scientifique suite à une importante éclosion en Amérique du Sud. Ce virus, peu étudié auparavant, peut causer des pathologies parfois graves chez les personnes infectées. Nous avons démontré que le virus Zika peut infecter les macrophages dérivés de monocytes. Plusieurs souches virales peuvent infecter les macrophages, mais certaines différences existent entre ces souches quant au taux d’infection et la cinétique de réplication. La réplication virale serait localisée autour du noyau de la cellule, comme ce qui est connu des autres membres de la famille des Flaviviridae. Les macrophages sont résistants aux effets cytopathiques du virus et peuvent donc jouer un rôle de réservoir viral à long terme. L’autophagie est un mécanisme nécessaire et important à la complétion du cycle viral du virus Zika dans les macrophages. Lors d’une co-infection entre le virus Zika et le VIH-1 dans les macrophages, il n’y a pas d’effet observé sur le taux d’infection et la réplication virale du VIH-1. Cependant, il y a une diminution de la réplication virale du virus Zika lors de ces co-infections. L’ajout de B18R, un inhibiteur de la réponse interféron de type I vient contrecarrer cette modulation, démontrant le mécanisme d’action de cette modulation. L’ajout d’un inhibiteur de la signalisation de la voie de l’interféron permet d’augmenter le taux d’infection des macrophages par le virus Zika. En résumé, le virus Zika peut infecter les macrophages et ces cellules pourraient être un réservoir viral à long terme, le virus a besoin de l’autophagie pour se répliquer et la production d’interféron de type I par les cellules qui sont infectées par un autre virus a un effet important sur la diminution de la réplication du virus Zika. / Zika virus is a pathogenic agent who recently gathered attention from the scientific community following a major outbreak in South America. This virus, who received little attention prior to this outbreak, can induce major pathologies in infected individuals. Our results indicate that Zika virus can infect monocyte-derived macrophages. Many viral strains can infect macrophages, but some differences were put in evidence, particularly regarding the infection rate and replication kinetic of the different strains. Viral replication occurs around the nucleus, which is expected and known from other viruses of the Flaviviridae family. Macrophages are resistant to the cytopathic effect of the virus, and can produce virus for many days. Autophagy is a necessary, and important cellular mechanism in completion of the virus life cycle. In the context of confections with Zika virus and HIV1, there is no effect observed in the infection rate and replication level of HIV-1. However, there is a decrease observed in the overall replication of Zika virus. The presence of B18R, a type I interferon inhibitor, restores a normal viral replication, demonstrating the mechanism behind this modulation. Addition of an inhibitor targeting the interferonmediated signalling pathway increases the infection rate of macrophages by Zika virus. In summary, Zika virus can infect macrophages, requires the autophagy signalling pathway to successfully replicate in macrophages, and is negatively impacted by interferon produced by its host cells infected by another virus.

Coronary artery disease (CAD) is the leading cause of death worldwide. Genome-wide association studies (GWAS) have identified more than 200 loci associated with CAD. Here, we investigated the functional effects of a locus tagged by rs61969072 (T/G), with the common allele (T) associated with protection from CAD. Expression quantitative trait loci (eQTL) analysis demonstrated a strong association between rs61969072 and CARS2 gene expression, which increased with the T allele, in various human tissues. CARS2 encodes the mitochondrial cysteinyl-tRNA synthetase, an enzyme that attaches cysteine to its cognate tRNA. We hypothesized that CARS2 is a candidate causal gene and that CARS2 confers a protective effect against CAD. We characterized CARS2 expression in macrophages and demonstrated decreased expression in pro-inflammatory M1 macrophages. Gene expression profiling following CARS2 siRNA knockdown revealed increased levels of several pro-inflammatory cytokines. Functional enrichment analysis identified the anti-inflammatory IL-10 signaling pathway, and western blotting showed that CARS2 attenuated IL-10 pathway activation through STAT3 phosphorylation. We also demonstrated that macrophage CARS2 knockdown in a macrophage/smooth muscle cell (SMC) co-culture model elicited gene expression changes indicative of a less contractile, pro-inflammatory, SMC phenotype. We then performed an in-depth analysis of differentially expressed genes following CARS2 knockdown. Several inflammatory pathways and functions were affected, particularly Protein Kinase R (PKR), implicated in Interferon Induction and Antiviral Response. Downstream of PKR is the NF-κB signaling pathway; CARS2 knockdown led to increased NF-κB protein expression but not activation, as measured by a luciferase reporter assay. Finally, we investigated potential mitochondrial mechanisms that could lead to inflammation. Reduced CARS2 levels were found to decrease mitochondrial membrane potential. However, there was a decrease in reactive oxygen species (ROS) levels and no changes in mitochondrial DNA release, metabolism, or mitochondrial bioenergetics. While ROS are often considered harmful due to their role in oxidative damage and inflammation, studies have shown that under certain contexts, ROS can have protective effects. Further studies are required to understand the mechanisms underlying the anti-inflammatory effects of CARS2. Overall, my findings highlight a novel anti-inflammatory role of CARS2 in human macrophages, consistent with the CAD protective effect of a common GWAS-identified variant.

BACKGROUND: Subarachnoid Hemorrhage (SAH) accounts for 3-5% of total stroke patients annually. Despite its rare incidence, SAH carries a 50% mortality rate. Survivors are often left with varying degrees of disability, many will never return to their previous jobs and require long-term care. One of the leading causes for this high mortality and morbidity rate in SAH is Delay Cerebral Ischemia (DCI). Researchers are now beginning to investigate neuroinflammation as the underlying cause for DCI. Studies have shown the activation of the innate immune system in the central nervous system is initiated by excess hemoglobin in the subarachnoid space. This process is mediated by the Toll-Like Receptor 4 expressed on the tissue-resident macrophages. Activated macrophages release pro-inflammatory cytokines and cause neuronal apoptosis in the surrounding tissue. However, macrophages may also mediate neuroprotection in SAH. A macrophage surface receptor called CD163 is responsible for the recognition and endocytosis of excess hemoglobin. The thesis provides a closer assessment of the neuroprotective role of macrophages in SAH patients. METHODS: Cerebrospinal fluid (CSF) was obtained from twenty three patients diagnosed with SAH (on day 1 and day 7 post-admission) or unruptured aneurysms. Immune cells were separated from CSF and analyzed by flow cytometry. The following antibody panel was used in this study: PE-anti-CD163, PeCy7-anti-CD15, and APC-anti-CD14. Macrophage expression of CD14 and CD163 was quantified using FlowJo. SAH patients were graded by the Hunt and Hess scale for the clinical states upon admission; modified Fisher scale for the size of the hemorrhage; and modified Rankin scale for clinical outcome at the time of discharge. RESULTS: Significant increase in macrophage CD14 and CD163 expression is observed in day 1 SAH patients (p<0.05) as compared to the control group. Male SAH patients have equivocal macrophages CD163 expression on day 1 as compared to the control group (p>0.05), and significantly higher expression on day 7 as compared to day 1(p<0.05). Female SAH patients have significantly higher macrophages CD163 expression on day 1 as compared to control patients (p<0.05), but slightly decreased expression on day 7 as compared to day 1(p>0.05). Lower macrophages CD163 expression is observed in SAH patients with more severe hemorrhage (marked by higher modified Fisher score), but not in patients with more severe clinical states at admission (marked by higher Hunt and Hess score). Furthermore, SAH patients with low day 1 macrophage CD163 expression and low expression on day 7 may be correlated with better clinical outcome (marked by lower modified Rankin score). However, more patients are required before correlation can be established. CONCLUSION: The data further support our previous findings in mouse SAH model that macrophages in the central nervous system may mediate inflammation via the increased expression TLR4, measured by increased expression of its co-receptor CD14. Macrophages also may be neuroprotective, mediated by increased expression of CD163 in SAH patients. The macrophage CD163 expression may be the key in determining clinical outcome in SAH patients, but additional patients are required to establish statistical significance. / 2017-06-16T00:00:00Z

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