L’athérosclérose est un phénomène inflammatoire caractérisé par un dépôt sur les artères de macrophages gorgés de cholestérol également appelés cellules spumeuses. Actuellement, trois principales molécules membranaires sont décrites comme assurant la sortie du cholestérol libre, dont l’ABCA1 (ATP Binding Cassette A1) qui joue un rôle physiologique important. Le but de ce travail est d’étudier l’impact de l’incorporation d’acides gras ω3 et celui de LDL modifiées sur la composition des phospholipides membranaires et d’établir des liens entre la modification de composition des phospholipides et l’altération du fonctionnement de l’ABCA1. Une étude lipidomique a été menée, en utilisant des méthodes de chromatographie couplées à la spectrométrie de masse. Les analyses ont été réalisées au moyen d’un système de chromatographie liquide en phase normale (NP-LC) permettant la séparation des différents phospholipides. La détection a été réalisée à l’aide de spectromètres de masse (MS) basse et ultra-haute résolution, ainsi que d’un détecteur à aérosol chargé. La chromatographie en phase gazeuse permet de connaître les proportions relatives d’acides gras. Ces techniques ont été utilisées pour réaliser une étude sur les paramètres de prétraitement (en amont du traitement des signaux analytiques), de décrire le lipidome de différents types de macrophages et d’établir les modifications de composition des phospholipides lors d’ajout chronique d’acides gras ω3 et/ou de LDL modifiées. Les résultats obtenus ont permis de définir une méthode de prétraitement de données LC-MS, d’évaluer la composition de trois types de macrophages et de modéliser les variations de l’efflux du cholestérol avec les modifications phospholipidiques observées pour les macrophages non spumeux. A partir de ces éléments, des thématiques communes, chimie biologie, ont pu aussi être identifiées comme l’analyse de «l’oxylipidome». / Atherosclerosis is an inflammatory disease characterized by a deposit on the arteries of macrophages full of cholesterol also called foam cells. Currently, three main membrane molecules are described as ensuring the release of free cholesterol, of which ABCA1 (ATP Binding Cassette A1) plays an important physiological role. The purpose of this work is to study the impact of the incorporation of ω3 fatty acids and modified LDL on membrane phospholipids composition and to establish the relationship between modification of the phospholipid composition and functionality of ABCA1. A lipidomic study was conducted using chromatographic methods coupled with mass spectrometry. Analyzes were carried out using normal phase liquid chromatography (NP-LC) allowing the separation of the different phospholipid classes. Detection was performed using low and ultra-high resolution mass spectrometers (MS) and a charged aerosol detector. Gas chromatography is used to determine fatty acids proportions. These techniques were used to study pretreatment parameters, to describe different macrophages lipidome and to establish phospholipid modifications during ω3 fatty acids chronic addition and / or modified LDL. The results obtained made it possible to define a method for LC-MS data pretreatment, to evaluate the composition of three types of macrophages, to model the variations of cholesterol efflux with the phospholipid modifications observed for non-foamy macrophages. From these elements, common themes, chemistry biology, could also be identified as the analysis of "oxylipidome".

Indiana University-Purdue University Indianapolis (IUPUI) / Fanconi anemia (FA) is a genetic disorder characterized by bone marrow (BM) failure. Previous studies suggest that FA patients exhibit alterations in immunologic function. However, it is unclear whether the immune defects are immune cell autonomous or secondary to leucopenia from evolving BM failure. The aim of the current study was to determine whether FA type C deficient (Fancc-/-) macrophages exhibit impaired function and contribute to an altered inflammatory response. In this study, primary peritoneal macrophage function and the inflammatory response of Fancc-/- immune cells after in vivo intraperitoneal (IP) administration of lipopolysaccharide (LPS) were assessed. Fancc-/- peritoneum exhibit normal macrophage distribution at baseline. However, Fancc-/- macrophages exhibit reduced adhesion both on fibronectin and endothelial cells, impaired migration toward monocyte chemotactic protein-1 (MCP-1) and macrophages-colony stimulating factor (M-CSF), and altered phagocytosis of E.coli and ImmunoglobulinG (IgG)-labeled latex beads compared to WT. An altered F-actin reorganization and impaired activation of RhoA were observed in Fancc-/- macrophages. After single LPS injection IP, Fancc-/- mice exhibited decreased macrophage recruitment, reduced peripheral inflammatory monocytes and impaired myeloid colony formation in presence of M-CSF. Upon M-CSF stimulation, Fancc-/- BM derived macrophages (BMDM) showed a decreased phosphorylation of AKT and ERK compared to WT, leading to reduced proliferation. Collectively, these data suggest that Fancc-/- macrophages and subsequent defects in adhesion, migration, phagocytosis, and recruitment in vivo. These data also support a Fancc-/- macrophage cells autonomous defect predisposing to an altered inflammatory response.

Recent evidence has shown the important role of macrophages in both tumor development and progression. To investigate the role of macrophages we used a mouse model known as MaFIA (Macrophage Fas Induced Apoptosis) mice that allows for the selective deletion of macrophages. Mice were given melanoma cells at various stages of depletion. Tumor mass was measured and organs were processed for flow cytometry to measure melanoma cell migration. The results show that mice receiving depletion treatment have larger tumor sizes and weights than those mice retaining their macrophage population. We detect metastasis in both the lung and kidney in both macrophage depleted and non depleted mice. The more macrophages in an organ the larger the amount of melanoma positive cells are detected.

Systemic sclerosis (SSc), or scleroderma, is a complex, rare disease of unknown etiology. Macrophages constitute a large portion of the immune cell infiltrate in the skin of patients with SSc, and are an important target of study. Particularly, the M2 macrophage has been implicated scleroderma and other fibrotic diseases as a key contributor to fibrotic processes. However, the definition of an M2 macrophage appears to change with context, and is poorly elucidated in different species. With varying characterizations between species and disease models, there is a need to establish some consensus on how to identify this macrophage in an uniform manner across species. We used a bioinformatic approach to identify a unique gene signature for the M2 macrophage phenotype, which is shared between human and mouse systems. We were able to confirm a 7-gene subset of this theorized signature using human and mouse in vitro systems. In addition, we selected one of the identified genes, Clec7a, and characterized its expression at the protein level on different macrophage phenotypes, across several human and mouse models. Our data show that Clec7a is a more selective marker of murine M2 macrophages than current reference markers, and is useful in human models as well. Using our M2-specific gene signature, we also identified a potential inhibitor of the signature and showed its effects on M2 marker expression. Finally, we showed some preliminary work into Clec7a expression in skin tissue from patients with scleroderma. Overall, our data suggest that Clec7a may be a valuable addition to the panel of markers used to characterize M2 macrophages and distinguish between macrophage phenotypes, and perhaps provide clarity into the development and function of the M2 macrophage. Better understanding of the M2 macrophage would ultimately be useful to the study of fibrotic diseases such as scleroderma, wherein this macrophage phenotype may be a viable target for antifibrotic therapy. / Thesis / Master of Science (MSc)

Bacterial mastitis is a significant problem for the dairy industry. A vaccine against mastitis pathogens could potentially target dendritic cells (DC). While there has been some research describing bovine DC populations in-vitro, little is known about DC in mammary tissue. In this study, immunohistofluorescence was used to identify and localize bovine mammary DC. DC were found in alveoli, in epithelia, and in interalveolar tissue. Fluorescence-activated cell sorting (FACS) was used to characterize mammary DC as expressing CD11c, MHC-II, CD205, CD11b, and CD8α. FACS allowed us to distinguish DC (CD14lo) from macrophages (CD14hi). Two DC subsets, CD11a-, CD11alo, were evident in the mammary gland while an additional CD11ahi population was identified in the supramammary lymph node. After phagocytosis of bacterial components such as lipopolysaccharide (LPS), DC undergo a maturation process, in which they upregulate homing receptors, such as CCR7, and antigen presentation markers, including MHCII and CD80. A primary cell culture model was used to evaluate changes in transcription of CD80 and CCR7 after LPS stimulation. Cell cultures contained digested and Ficoll separated mammary tissue or supramammary lymph node tissue. While the presence of CCR7 and CD80 was confirmed, CD80 and CCR7 transcripts were not upregulated after LPS stimulation. Further, CD11c, CD14, MHCII, CD11b, CD11a, and CD205 protein levels, as assessed by FACS, were similar in LPS stimulated cultures and unstimulated controls. Overall, these studies provide a better understanding of mammary gland immunology, while potentially aiding in the development of novel DC based vaccines. / Master of Science

No description available.

The virulence of the opportunistic fungal pathogen Candida albicans is in part due to its ability to switch between a yeast and hyphal form; permitting physical rupture and escape from macrophages after phagocytosis. This interesting feature makes C. albicans a good model organism to study in the context of phagosome maturation and in particular, the role of the small GTPase Rab14 in this process. Rab14 is recruited to bacterial phagosomes containing Chlamydia trachomatis (Capmany & Damiani, 2010) and Mycobacterium bovis BCG (Kyei et al, 2006) and aids their survival in macrophages but its role in phagosomes containing fungal pathogens is not known. Here, an important role for Rab14 in protecting macrophages against hyphal mediated lysis by C. albicans is demonstrated. Macrophages were transfected to express eGFP-Rab14, or dominant negative variants (eGFP-Rab14 S25N and eGFP-Rab14N124I); or were transfected with anti-Rab14 siRNA; then infected with live C. albicans and observed using sophisticated live cell imaging and analysis tools. Phagosomes containing live C. albicans became transiently Rab14-positive within 2 minutes following engulfment. Interestingly, a prolonged retention of Rab14 on phagosomes depended on C. albicans morphology. Phagosomes containing hyphal forms retained Rab14 for twice as long as for phagosomes containing the yeast form. Depletion of endogenous Rab14 did not affect macrophage migration towards C. albicans, the rate of engulfment or acquisition of markers of early phagosome maturation to phagosomes containing C. albicans. Furthermore, reduced Rab14 expression did not influence the kinetics of Rab5 localisation to phagosomes in macrophages that were co-transfected. Importantly, partially depleting Rab14 delayed the appearance of late phagosome maturation indicators LAMP1 and activated cathepsin in phagosomes containing live C. albicans.Rab14 knockdown was associated with a significant increase in macrophage killing by C. alibica The data presented in this thesis demonstrate the dynamic relationship between host and pathogen which can be visualised in real time at the level of individual phagosomes. Rab14 plays an important role during phagosome maturation which impacts on the later stages of phagosome maturation and is important for phagocyte survival after phagocytosis of C. albicans.

published_or_final_version / abstract / toc / Pharmacology / Master / Master of Philosophy

No description available.

The cellular origin of prostaglandins in human tumors was investigated using cell fractionation procedures and high resolution gas chromatography. Additionally, the role of macrophages and prostaglandins on human tumor cloning in vitro was investigated. Spontaneous human tumors were prepared as single cell suspensions which were subsequently manipulated to yield macrophage-enriched, and tumor cell enriched (macrophage-depleted) subpopulations of cells. A fused silica capillary gas chromatographic analysis with electron capture detection was developed to measure derivatized prostaglandins in the supernatant of the cell subpopulation incubations. The derivative used for the analysis was the pentafluorobenzyl ester-methoxime-trimethyl-silyl ether. The assay showed a detection limit of 25 picograms of prostaglandins E₁, E₂, F₂ₐ, and I₂ (which was detected as 6-Keto-PGF₁ₐ). Analysis of cell subpopulations of seventeeen tumor samples showed that the macrophage-enriched cells were responsible for the large majority of prostaglandin production in vitro (p ≤ 0.02). It was found that the major prostaglandins were PGE₂ and PGI₂. The range of values measured for macrophage produced prostaglandin E₂ was 1.1 to 704.8 ng/ml after a 24 hour incubation of 10⁶ cells. Prostaglandin I₂ was also produced by the macrophage-enriched cell subpopulation with values ranging from 1.2 to 334.3 ng/ml. This is the first report of prostaglandin I₂ production by host macrophages infiltrating human tumors. Studies of the effect of macrophage depletion and reconstitution on the ability of tumor cells to form colonies in vitro were performed. A two layer soft agar assay was used to evaluate tumor cells clonogenicity. The results demonstrated that macrophages infiltrating human carcinoma samples function in a supportive role for tumor cell colony formation in vitro. Using a prostaglandin synthesis inhibitor, flurbiprofen, it was shown that this support was not the result of a direct effect of prostaglandins on the tumor cells. Possible roles for macrophage produced prostaglandins in cancer are discussed.