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Phagocytosis is the process by which cells engulf foreign bodies. It is the hallmark behavior of white blood cells, being the process through which those cells ingest and degrade pathogens and debris. To date a large amount of research has focused on documenting the existence and role of biochemical components involved with phagocytosis. Scores of signaling molecules have been implicated in the complex signal cascade which drives the process. These molecules are small (typically no larger than 5 nanometers) and operate in a crowded, chemically “noisy,” environment, yet they coordinate the cell's activity over comparatively expansive distances (as large as 20 micrometers). How these molecular processes scale-up to coordinate the activities of the cell over such massive distances is largely unknown. Using a planar analog of phagocytosis termed “frustrated phagocytosis,” we experimentally demonstrate that phagocytosis occurs in three distinct phases: initial cell-antigen binding, symmetric spreading, and late-stage contraction. Initial binding and symmetric spreading appears to be both mechanically and chemically similar to the quasi-universal cellular behaviors of adhesion and migration. Adhesion and migration have received extensive attention from the biophysics community in recent years. Leveraging these similarities, we adapt the biomechanical frameworks used in models of migration to phagocytosis. We show that macroscopic properties such as a cell's effective viscosity and membrane cortical tension can be used to model cell behavior during phagocytosis. Our experiments reveal that late-stage contraction distinguishes frustrated phagocytosis from other spreading behaviors. This contraction is myosin dependent. Additionally we demonstrate, for the first time, that late-stage contraction corresponds with formation of a contractile F-actin belt. Based on the dynamic contraction model (DC) developed to explain actin structure during cell migration we propose a DC model of phagocytosis which posits that contractile belt formation is the result of a late-stage myosin activity coupled with F-actin.

O aumento dos casos de diarreia causados por EPECa evoca a sua capacidade de adaptação e patogenicidade. O objetivo deste estudo foi investigar o comportamento da EPECa na interação com macrófagos (fagocitose e antifagocitose). O estudo da fagocitose das cepas LB7 (O55:H7), LB13 (O111:abH9) e BA487 (O55:H7) em macrófagos J774A1 mostrou sobrevivência intracelular em presença de NO. EPECa impede a maturação do vacúolo parasitóforo. A sobrevivência em macrófago derivado de C3H/HeJ, mutante do tlr4, foi reduzida e em macrófago de C57BL/6 não foi observada. O fator antigagocítico (Fa) secretado pela LB7, já detectado pelo grupo, apresenta natureza peptídica e a sua ação não é específica de EPEC, pois inibe também a fagocitose de Shigella e látex. A secreção do Fa foi avaliada em M9 e DMEM. O produto do fracionamento do sobrenadante do cultivo por SPE apresentou Fa em ambos meios. No entanto, a secreção em M9 é baixa e não foi detectada por HPLC. O Fa do DMEM obtido por HPLC mostrou-se citotóxico. Novos meios de cultivo deverão ser estudados para a identificação do Fa. / The increase in the numbers of diarrhea cases caused by aEPEC denotes its adaptability and pathogenicity. The objective of this study was to investigate the behavior of aEPEC in the interaction with macrophages (phagocytosis and anti-phagocytosis). Strains LB7 (O55:H7), LB13 (O111:abH9) and BA487 (O55:H7) were shown to survive within J774A1 macrophages in the presence of NO. aEPEC prevents maturation of the parasitophorus vacuole. Survival inside C3H/HeJ derived macrophages, mutant for tlr4, decreased and was not observed in C57BL/6 derived macrophages. The anti-phagocytic factor (AF), secreted by LB7 and previously detected by our group, is peptidic and its action is not specific to EPEC as it also inhibits phagocytosis of Shigella and latex. Secretion of AF was evaluated in M9 and DMEM. AF was detected after SPE fractionation of both culture media. However, the secretion in M9 is low and was not detected by HPLC. The DMEM HPLC fraction containing AF was cytotoxic. New culture media will be studied for the identification of AF.

Within the paradigm of HIV-1 infection, macrophages play a crucial role as long-lived viral reservoirs. However, cell-free virus infection is inefficient and is unlikely to explain the levels of infection observed in vivo. To investigate the hypothesis that macrophages might be infected via direct contact with HIV-1-infected T cells, macrophage and HIV-1-infected T cell cocultures were imaged in real time. I observed that macrophages preferentially phagocytosed HIV-1-infected T cells and, using long-term culture assays, I established that following coculture the macrophage became productively infected. Phagocytosis of HIV-1-infected cells occurred independently of viral tropism; however, productive infection following T cell phagocytosis was restricted by viral tropism. Imaging flow cytometry showed that macrophages primarily phagocytose dying HIV-1-infected T cells. However, a significant population of HIV-1-infected 'healthy' cells were also taken up. Furthermore, ICAM-1 was identified as mediating the uptake of HIV-1-infected T cells. These results indicate that apoptosis plays a significant, but not sufficient, role in the mechanism for recognition and uptake of HIV-1-infected T cells. The response of macrophages to HIV-1 infection remains controversial. Using both primary macrophages and a monocyte/macrophage NFκB reporter line assay, I demonstrated that macrophages are activated in response to HIV-1-infected T cells. In addition, during coculture with HIV-1-infected T cells, macrophages upregulated secretion of Th1 cytokines, with associated dysregulation of regulatory cytokines. Finally, data presented suggest that polarisation of macrophages towards M1 and M2 phenotypes alters the susceptibility to HIV-1 infection in the cell-to-cell route.

Red blood cells (RBCs) are vital for oxygen delivery to tissues and constitute the vast majority of all cells in blood. After leaving the red bone marrow as mature cells, RBCs have a lifespan of approximately 120 days before they are removed from the circulation by macrophages, mainly in the spleen and liver. RBC transfusion is a common therapy in modern healthcare. Major surgery, numerous cancer treatments and other, often lifesaving, interventions would be unthinkable without available blood supply. For this reason, hospitals store donated RBCs in blood banks. The metabolic and structural changes that occur during prolonged storage of RBCs (the storage lesion) have been studied in detail in vitro and include oxidative stress, a reduction in glycolysis, increased membrane rigidity and shedding of microparticles from the RBC membrane. Stored RBCs share several features of senescent RBCs, but also with RBCs undergoing an apoptotic-like process called eryptosis. A consequence of the storage lesion is the fact that as much as 25% of stored RBCs could be rapidly removed from the circulation within 24 hours after transfusion. The mechanisms behind this rapid macrophage-mediated recognition and removal of stored RBCs, and its immunological consequences, remain largely unknown. Therefore, the aims of this thesis were to investigate if cryopreserved human RBCs induced an inflammatory response following autologous transfusion into healthy volunteers, and to further understand the mechanisms behind macrophage recognition of stored RBCs in vitro and in vivo. Autologous transfusion of two units of cryopreserved RBCs into healthy human recipients was found to be associated with an increased extravascular RBC elimination already at 2 hours after transfusion. However, there were no signs of an increased production of any of the investigated pro-inflammatory cytokines, indicating that an increase in the destruction of RBCs per se did not induce an inflammatory response. Eryptosis is a form of induced RBC death associated with an increased cytoplasmic Ca2+ uptake. We found that a subset of human RBCs increased their Ca2+ permeability during prolonged storage at +4°C. Using a murine model, to further understand how RBCs with an increased Ca2+ permeability were eliminated by phagocytic cells in the spleen, it was found that such RBCs were taken up by marginal zone macrophages and dendritic cells (DCs) in a manner distinct from that of naturally senescent RBCs. The DC population particularly efficient in this process expressed CD207 and are known for their ability to promote immunological tolerance. Eryptotic cell uptake was not regulated by the phagocytosis-inhibitory protein CD47 on the RBCs. To investigate how RBCs damaged during liquid storage are recognized and taken up by macrophages, a model to store and transfuse murine RBCs was developed. This storage model generated murine RBCs with several characteristics similar to that of stored human RBCs (i.e. loss of ATP, formation of RBC microparticles and rapid clearance of up to 35% of the RBCs during the first 24 h after transfusion). In vitro phagocytosis of human as well as murine stored RBCs was serum dependent and could be inhibited by blocking class A scavenger receptors using fucoidan or dextran sulphate. In conclusion, the findings of this thesis contribute to further understanding how changes inflicted to RBCs during storage direct the fate of these cells in their interaction with cells of the immune system after transfusion. The observation of an increased Ca2+ permeability of stored RBCs, and the possible recognition of such cells by tolerance-promoting DCs, in combination with the findings that class A scavenger receptors and serum factors may mediate recognition of stored RBCs, may result in novel new directions of research within the field of transfusion medicine.

The rate limiting step of phagocytosis is the binding of collagen to specific receptors, which include β1 integrins and the discoidin domain receptor 1 (DDR1). While these two receptors may interact, the functional nature of these interactions is not defined. We examined the effects of DDR1 over-expression on β1 integrin function and determined that DDR1 over-expression enhanced cell attachment through β1 integrins. These data are consistent with data showing that DDR1 over-expression enhanced cell-surface, but not total, β1 integrin expression and activation. As shown by experiments with endoglycosidase H, DDR1 over-expression increased glycosylation of the β1 integrin subunit. Collectively these data indicate that DDR1 enhances β1 integrin interactions with fibrillar collagen, possibly by affecting the processing and trafficking of β1 integrins to the cell surface. Our data provide insight into the mechanisms by which fibrotic conditions such as cyclosporine A-induced gingival overgrowth are regulated.

The rate limiting step of phagocytosis is the binding of collagen to specific receptors, which include β1 integrins and the discoidin domain receptor 1 (DDR1). While these two receptors may interact, the functional nature of these interactions is not defined. We examined the effects of DDR1 over-expression on β1 integrin function and determined that DDR1 over-expression enhanced cell attachment through β1 integrins. These data are consistent with data showing that DDR1 over-expression enhanced cell-surface, but not total, β1 integrin expression and activation. As shown by experiments with endoglycosidase H, DDR1 over-expression increased glycosylation of the β1 integrin subunit. Collectively these data indicate that DDR1 enhances β1 integrin interactions with fibrillar collagen, possibly by affecting the processing and trafficking of β1 integrins to the cell surface. Our data provide insight into the mechanisms by which fibrotic conditions such as cyclosporine A-induced gingival overgrowth are regulated.

Apoptosis is a form of programmed cell death that is essential in such processes as organ and tissue remodeling and maturation of hematopoietic cells. The clearance of apoptotic cells is essential to prevent autoimmune responses to sequestered antigens. This process is mediated by phagocytes of the monocyte lineage. Before phagocytosis can occur, macrophages must be recruited to the apoptotic cells through chemotaxis. Products of the reaction catalyzed by the phospholipases A2 (PLA2) have been shown to induce monocyte chemotaxis either directly or indirectly. Some investigators have implicated a cytosolic calcium-independent PLA2 (iPLA2) in the production of these products during apoptosis. However, a recent report suggests that the secreted group IIa (sPLA2) binds to surfaces of apoptotic cells. The "receptor" for this pool of sPLA2 is the rod domain of vimentin, an intermediate filament protein that is exposed by caspase activity when cells undergo apoptosis. Based on these observations, we hypothesize that the exposure of vimentin on apoptotic cells traps a pool of catalytically active sPLA2 that then generates the bioactive lipids that induce macrophage chemotaxis. In our methods, [3H]-oleate labeled E-coli is used as a substrate for sPLA2 and enzyme activity is quantified by scintillation counting of released radiolabeled oleic acid. Apoptosis is induced with anti-fas (CD-95) on Jurkat cells and monitored through annexin-V binding and propidium iodide(PI) staining followed by flow cytometric analyses. THP-1 monocytes are employed in chemotaxis assay with monocyte chemotactic protein (MCP-1) as a positive control. The preliminary data show equivalent group IIa sPLA2 association with anti-fas treated and control cells, and the enzyme remains catalytically active when bound. In line with the hypothesis, trapped sPLA2 generated soluble molecules that directly or indirectly induced migration of THP-1 monocytes. However, the similar binding effect observed with apoptotic or control cells is surprising and experiments are being planned to determine if the interaction between IIa sPLA2 and apoptotic cells is vimentin dependent.

Le système de l’immunité innée cérébrale (SIIC) est principalement composé des cellules microgliales. En réponse à des stimuli immuns, inflammatoires ou un trauma neurologique, la microglie s’active et produit des facteurs pro et anti-inflammatoires qui d’une part coordonnent la réponse de l’immunité innée cérébrale et d’autre part modulent l’activité neuronale et, in fine, le comportement. Plus récemment, les cellules microgliales se sont révélées jouer un rôle clé dans le développement cérébral. Ainsi, par leurs activités de phagocytose, elles participent à la maturation des réseaux neuronaux. Si l’activation du SIIC permet de défendre le tissu cérébral des agressions, l’activation prolongée des cellules microgliales a aussi des effets délétères. Ainsi, dans le cerveau adulte, la production soutenue de cytokines inflammatoires contribue au développement de pathologies neurodégénératives. Au cours du développement les stimuli inflammatoires, en perturbant l’activité des cellules microgliales conduisent à une dysfonction de circuits neuronaux qui pourrait être impliquée dans des pathologies neuropsychiatriques à composante neurodéveloppementale. La compréhension de la régulation des cellules microgliales et de leur réponse est donc capitale. L’activité microgliale repose sur ses propriétés morphologique, dynamique et sa communication avec les neurones qui impliquent des profils de synthèse de facteurs (cytokines, chemokines, facteurs de croissance, etc..) et de récepteurs particuliers, la polarisation vers un phénotype pro ou anti-inflammatoire et la phagocytose. Peu d’études ont caractérisé l’ensemble des propriétés morphofonctionnelles des cellules microgliales in vivo. Par la combinaison d’approches de FACS, immunohistochimie, microscopie confocale et reconstruction en 3D, microscopie bi-photonique et dosage des facteurs de communication, il est aujourd’hui possible de mieux caractériser ces cellules afin de comprendre leur régulation par l’environnement et l’impact (bénéfique ou délétère) sur les fonctions neuronales. L’objectif général de cette thèse a été d’étudier les propriétés morphofonctionnelles des cellules microgliales in vivo dans deux situations physiopathologiques, une inflammation induite par l’administration périphérique de lipopolysaccharide (LPS) et une déficience alimentaire en acides gras polyinsaturés (AGPI) de type n-3, connus pour leurs propriétés immunomodulatrices. La première étude nous a permis de développer des outils nécessaires à l’étude de la plasticité morphofonctionnelle de la microglie et d’apporter de nouveaux éléments de compréhension de l’impact d’une inflammation périphérique sur l’activité de ces cellules in vivo. Dans la deuxième partie de cette thèse, nous avons montré pour la première fois que le statut alimentaire maternel en AGPI n-3 influence les propriétés morphofonctionnelles des cellules microgliales au cours du développement post-natal ainsi que l’activité des réseaux neuronaux. De façon générale, nos résultats apportent des éléments de compréhension des relations entre plasticité morphologique et fonctionnelle des cellules microgliales in vivo. / The brain innate immune system is mainly composed of microglial cells. Microglia are activated in response to an immune or inflammatory stimuli or a trauma, and then produce pro- and anti-inflammatory factors. These factors drive the innate immune response and can modulate neuronal activity and in fine, learning and memory. Recently, microglia have been shown to play a key role during brain development. Via their phagocytic activity, microglial cells can participate to neuronal networks maturation. Although brain innate immune system defends brain tissue from aggression, chronic activation of microglia can also be deleterious. In the adult brain, chronic production of inflammatory cytokines can contribute to the pathogenesis of neurodegenerative diseases. During development, inflammatory stimuli modifying microglia activity and homeostasis could lead to neuropsychiatric diseases with a neurodevelopmental origin. Understanding how microglia are regulated and how they respond to various stimuli is therefore crucial.Microglia activity is characterized by morphological and dynamic properties of microglia,by its communication with neurons by its polarization into a specific phenotype, and by their phagocytic profile. Few studies have characterized all the morphofunctional properties of microglial cells in vivo. Using a combination of approaches including FACS, immunohistochemistry, confocal microscopy, 3D reconstruction, two-photon microscopy and communication factors assays, it is now possible to better characterize these cells in order to understand their regulation by the environment and the resulting impact (beneficial or deleterious) on neuronal functions. The main goal of this thesis was to study the morphofunctional properties of microglial cells in vivo in two pathophysiological states: a peripheral inflammation induced by a peripheral injection of lipopolysaccharide (LPS) and in an n-3 PUFAs nutritional state. In the first study, we developed tools to investigate microglial morphofunctional plasticity and gained a better understanding of the impact of peripheral inflammation on the activity of these cells in vivo. In the second part of this thesis, we showed for the first time that maternal nutritional status in n-3 PUFAs affect the morphofunctional properties of microglial cells and the establishment of neural circuits during the postnatal development of the pups. Overall, our results provide new insights in the relationship between morphological and functional plasticity of microglial cells in vivo.