Régulation moléculaire de la barrière hémo-encéphalique

La Sclérose en plaques (SEP) est une maladie auto-immune inflammatoire démyélinisante du système nerveux central (SNC), lors de laquelle des cellules inflammatoires du sang périphérique infiltrent le SNC pour y causer des dommages cellulaires. Dans ces réactions neuroinflammatoires, les cellules immunitaires traversent le système vasculaire du SNC, la barrière hémo-encéphalique (BHE), pour avoir accès au SNC et s’y accumuler. La BHE est donc la première entité que rencontrent les cellules inflammatoires du sang lors de leur migration au cerveau. Ceci lui confère un potentiel thérapeutique important pour influencer l’infiltration de cellules du sang vers le cerveau, et ainsi limiter les réactions neuroinflammatoires. En effet, les interactions entre les cellules immunitaires et les parois vasculaires sont encore mal comprises, car elles sont nombreuses et complexes. Différents mécanismes pouvant influencer la perméabilité de la BHE aux cellules immunitaires ont été décrits, et représentent aujourd’hui des cibles potentielles pour le contrôle des réactions neuro-immunes. Cette thèse a pour objectif de décrire de nouveaux mécanismes moléculaires opérant au niveau de la BHE qui interviennent dans les réactions neuroinflammatoires et qui ont un potentiel thérapeutique pour influencer les interactions neuro-immunologiques. Ce travail de doctorat est séparé en trois sections. La première section décrit la caractérisation du rôle de l’angiotensine II dans la régulation de la perméabilité de la BHE. La seconde section identifie et caractérise la fonction d’une nouvelle molécule d’adhérence de la BHE, ALCAM, dans la transmigration de cellules inflammatoires du sang vers le SNC. La troisième section traite des propriétés sécrétoires de la BHE et du rôle de la chimiokine MCP-1 dans les interactions entre la BHE et les cellules souches. Dans un premier temps, nous démontrons l’importance de l’angiotensinogène (AGT) dans la régulation de la perméabilité de la BHE. L’AGT est sécrété par les astrocytes et métabolisé en angiotensine II pour pouvoir agir au niveau des CE de la BHE à travers le récepteur à l’angiotensine II, AT1 et AT2. Au niveau de la BHE, l’angiotensine II entraîne la phosphorylation et l’enrichissement de l’occludine au sein de radeaux lipidiques, un phénomène associé à l’augmentation de l’étanchéité de la BHE. De plus, dans les lésions de SEP, on retrouve une diminution de l’expression de l’AGT et de l’occludine. Ceci est relié à nos observations in vitro, qui démontrent que des cytokines pro-inflammatoires limitent la sécrétion de l’AGT. Cette étude élucide un nouveau mécanisme par lequel les astrocytes influencent et augmentent l’étanchéité de la BHE, et implique une dysfonction de ce mécanisme dans les lésions de la SEP où s’accumulent les cellules inflammatoires. Dans un deuxième temps, les techniques établies dans la première section ont été utilisées afin d’identifier les protéines de la BHE qui s’accumulent dans les radeaux lipidiques. En utilisant une technique de protéomique nous avons identifié ALCAM (Activated Leukocyte Cell Adhesion Molecule) comme une protéine membranaire exprimée par les CE de la BHE. ALCAM se comporte comme une molécule d’adhérence typique. En effet, ALCAM permet la liaison entre les cellules du sang et la paroi vasculaire, via des interactions homotypiques (ALCAM-ALCAM pour les monocytes) ou hétérotypiques (ALCAM-CD6 pour les lymphocytes). Les cytokines inflammatoires augmentent le niveau d’expression d’ALCAM par la BHE, ce qui permet un recrutement local de cellules inflammatoires. Enfin, l’inhibition des interactions ALCAM-ALCAM et ALCAM-CD6 limite la transmigration des cellules inflammatoires (monocytes et cellules T CD4+) à travers la BHE in vitro et in vivo dans un modèle murin de la SEP. Cette deuxième partie identifie ALCAM comme une cible potentielle pour influencer la transmigration de cellules inflammatoires vers le cerveau. Dans un troisième temps, nous avons pu démontrer l’importance des propriétés sécrétoires spécifiques à la BHE dans les interactions avec les cellules souches neurales (CSN). Les CSN représentent un potentiel thérapeutique unique pour les maladies du SNC dans lesquelles la régénération cellulaire est limitée, comme dans la SEP. Des facteurs qui limitent l’utilisation thérapeutique des CSN sont le mode d’administration et leur maturation en cellules neurales ou gliales. Bien que la route d’administration préférée pour les CSN soit la voie intrathécale, l’injection intraveineuse représente la voie d’administration la plus facile et la moins invasive. Dans ce contexte, il est important de comprendre les interactions possibles entre les cellules souches et la paroi vasculaire du SNC qui sera responsable de leur recrutement dans le parenchyme cérébral. En collaborant avec des chercheurs de la Belgique spécialisés en CSN, nos travaux nous ont permis de confirmer, in vitro, que les cellules souches neurales humaines migrent à travers les CE humaines de la BHE avant d’entamer leur différenciation en cellules du SNC. Suite à la migration à travers les cellules de la BHE les CSN se différencient spontanément en neurones, en astrocytes et en oligodendrocytes. Ces effets sont notés préférentiellement avec les cellules de la BHE par rapport aux CE non cérébrales. Ces propriétés spécifiques aux cellules de la BHE dépendent de la chimiokine MCP-1/CCL2 sécrétée par ces dernières. Ainsi, cette dernière partie suggère que la BHE n’est pas un obstacle à la migration de CSN vers le SNC. De plus, la chimiokine MCP-1 est identifiée comme un facteur sécrété par la BHE qui permet l’accumulation et la différentiation préférentielle de cellules souches neurales dans l’espace sous-endothélial. Ces trois études démontrent l’importance de la BHE dans la migration des cellules inflammatoires et des CSN vers le SNC et indiquent que de multiples mécanismes moléculaires contribuent au dérèglement de l’homéostasie du SNC dans les réactions neuro-immunes. En utilisant des modèles in vitro, in situ et in vivo, nous avons identifié trois nouveaux mécanismes qui permettent d’influencer les interactions entre les cellules du sang et la BHE. L’identification de ces mécanismes permet non seulement une meilleure compréhension de la pathophysiologie des réactions neuroinflammatoires du SNC et des maladies qui y sont associées, mais suggère également des cibles thérapeutiques potentielles pour influencer l’infiltration des cellules du sang vers le cerveauMultiple Sclerosis is an inflammatory demyelinating disease in which immune cells from the peripheral blood infiltrate the central nervous system (CNS) to cause a pathologic neuroinflammatory reaction. Blood borne leucocytes cross the restrictive cerebral endothelium, the blood brain barrier (BBB), to gain access to the CNS parenchyma and cause cellular damage leading to the characteristic demyelinating lesions. The BBB is the interface between the blood and the CNS and as such is a critical mediator of neuro-immune reactions and an important therapeutic target to modulate neuroinflammation. It is essential to have a better understanding of the molecular mechanisms that regulate the BBB properties to elaborate new therapeutic strategies to modulate the BBB and thus the local neuroinflammation reaction. This Ph.D. thesis describes three distinct molecular mechanisms which regulate key BBB properties. The first section describes a novel role for the renin-angiotensin system (RAS) in the neuro-vascular unit (NVU) as a regulator of paracellular permeability. The second part of this thesis characterises the role of a novel adhesion molecule of the BBB, ALCAM. The third part of this work studies the interactions between neural stem cells (NSC) and the BBB and identifies MCP-1 as a critical factor involved in NSC recruitment to the CNS. In the first experimental section we provide evidence that angiotensinogen (AGT) produced and secreted by astrocytes, is cleaved into angiotensin II (AngII) and acts on type 1 angiotensin receptors (AT1) expressed by BBB endothelial cells (ECs). Activation of AT1 restricts the passage of molecular tracers across human BBB-derived ECs through threonine-phosphorylation of the tight junction protein occludin and its mobilization to lipid raft membrane microdomains. We also show that AGT knockout animals have disorganized occludin strands at the level of the BBB and a diffuse accumulation of the endogenous serum protein plasminogen in the CNS, as compared to wild type animals. Finally, we demonstrate a reduction in the number of AGT-immunopositive perivascular astrocytes in multiple sclerosis (MS) lesions, which correlates with a reduced expression of occludin similarly seen in the CNS of AGT knockout animals. Such a reduction in astrocyte-expressed AGT and AngII is dependent, in vitro, on the pro-inflammatory cytokines tumor necrosis factor-α and interferon-γ. Our study defines a novel physiological role for AngII in the CNS and suggests that inflammation-induced downregulation of AngII production by astrocytes is involved in BBB dysfunction in MS lesions. In the second experimental part we focus on adhesion molecules of the BBB. Using a lipid raft-based proteomic approach, we identified ALCAM (Activated leukocyte cell adhesion molecule) as an adhesion molecule involved in leukocyte migration across the BBB. ALCAM expressed on BBB endothelium co-localized with CD6 expressed on leukocytes and with BBB endothelium transmigratory cups. ALCAM expression on BBB cells was up-regulated in active multiple sclerosis and experimental auto-immune encephalomyelitis (EAE) lesions. Moreover, ALCAM blockade restricted transmigration of CD4+ lymphocytes and monocytes across BBB endothelium in vitro and in vivo, and reduced the severity and time of onset of EAE. Our findings point to an important role for ALCAM in leukocyte recruitment into the brain and identify ALCAM as a potential therapeutic target to dampen neuroinflammation. The third experimental part of this thesis studies the interactions between NCS and BBB. NCS represent an attractive source for cell transplantation and neural tissue repair. After systemic injection, NCS are confronted with the specialized BBB endothelial cells before they can enter the brain parenchyma. We investigated the interactions of human fetal neural precursor cells with human brain endothelial cells in an in vitro model using primary cultures. We demonstrated that human fetal neural precursor cells efficiently and specifically migrate to sub-endothelial space of human BBB-endothelium, but not pulmonary artery endothelial cells. When migrated across BBB-endothelial cells, fetal neural precursor cells spontaneously differentiate to neurons, astrocytes and oligodendrocytes. Effective migration and subsequent differentiation was found to be dependant on the chemokine CCL2/MCP-1, but not CXCL8/IL-8. Our findings suggest that an intact blood-brain barrier is not an intrinsic obstacle to neural stem cell migration into the brain and that differentiation of neural precursor cells occur in a sub-endothelial niche, under the influence of the chemokine CCL2/MCP-1. These three experimental sections demonstrate the crucial roles that the BBB plays in regulating the CNS homeostasis. Under pathological conditions, such as during neuro-immune reactions, the BBB is altered and becomes an important local player. The three different molecular mechanisms described in this thesis, contribute to our understanding of the BBB and may allow for the development of novel therapeutic strategies to limit neuroinflammation

Study of class I and class II P-glycoprotein chimeras

The Multidrug drug resistance (MDR) phenotype is a major limitation to chemotherapy. The MDR phenotype arises when class I P-glycoproteins (P-gps) are overexpressed in cultured cells in vitro and in tumor cells in vivo. P-gps are encoded in rodents by three genes: mdr1, mdr2 and mdr3. Class I P-gps (Mdrl, Mdr3) confer drug resistance upon transfection in non-resistant cells and act as ATP dependent drug efflux pumps, class II P-gps (Mdr2) do not convey MDR but act as lipid flippases in the canalicular membranes of hepatocytes. P-gps are composed of twelve transmembrane domains and two nucleotide binding sites. The membrane spanning domains (MSD) of Pgp show the lowest level of similarity amongst family members and are postulated to be responsible for the different substrate specificities of the two classes of P-gp. In this study, chimeric cDNAs were constructed between class I and class II P-gps. The cDNAs were optimized for expression and study in yeast (Saccharomyces cerevisiae and Pichia pastoris) expression systems. The ATPase activity of the expressed constructs was studied by vanadate induced trapping of nucleotides

Medfly Ceratitis capitata Wiedemann (Dipt., Trypetidae) as a rot vector in laboratory conditions

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Isolation of human brain endothelial cells and characterization of lipid raft-associated proteins by mass spectroscopy

The blood-brain barrier (BBB) limits the movements of molecules, nutrients, and cells from the systemic blood circulation into the central nervous system (CNS), and vice versa, thus allowing an optimal microenvironment for CNS development and function. The brain endothelial cells (BECs) form the primary barrier between the blood and the CNS. In addition, pericytes, neurons, and astrocytes that make up the neurovascular unit support the BEC functions and are essential to maintain this restrictive permeability phenotype. To better understand the molecular mechanisms underlying BBB properties, we propose a method to study the proteome of detergent resistant microdomain, namely lipid rafts, from human primary cultures of BECs. This chapter describes a robust human BECs isolation protocol, standard tissue culture protocols, ECs purity assessment protocols, lipid raft microdomain isolation method, and a mass spectrometry analysis technique to characterize the protein content of membrane microdomains.Peer reviewed: YesNRC publication: Ye

ALCAM is a Novel Adhesion Molecule of the Inflammed Endothelium Involved in Leukocyte Trafficking to the Central Nervous System

Leukocyte trafficking from the blood to local inflammatory sites is essential for the initiation and maintenance of tissue specific immune responses. In autoimmune diseases such as multiple sclerosis (MS), leukocyte transmigration from the blood to the target organ is dependent on intercellular cell adhesion molecule 1 (ICAM-1) and vascular cell adhesion molecule 1 (VCAM-1) expressed by the endothelial cells (ECs). In this study we describe CD166/activated leukocyte cell adhesion molecule (ALCAM) as a novel adhesion molecule of the human blood\u2013brain barrier (BBB). We demonstrate that inflammatory cytokines up-regulate the expression of ALCAM on the surface of BBB-ECs in vitro and that endothelial ALCAM co-localizes with leukocyte expressed-CD6 in the transmigratory cup during migration. We further show that ALCAM blocking antibody restricts the transmigration of CD4, CD14 and CD19 immune cells across human BBB-ECs and that ALCAM expression is increased on ECs within active MS lesions, as compared to normal appearing white matter and to non-MS brains. These results suggest an important role for ALCAM in the recruitment of leukocytes to the CNS and identify ALCAM as a potential target to modulate CNS inflammatory reactions.Peer reviewed: YesNRC publication: Ye

A Loss-of-Function Mutation in the Integrin Alpha L ( Itgal ) Gene Contributes to Susceptibility to Salmonella enterica Serovar Typhimurium Infection in Collaborative Cross Strain CC042

International audienceSalmonella is an intracellular bacterium found in the gastrointestinal tract of mammalian, avian, and reptilian hosts. Mouse models have been extensively used to model in vivo distinct aspects of human Salmonella infections and have led to the identification of several host susceptibility genes. We have investigated the susceptibility of Collaborative Cross strains to intravenous infection with Salmonella enterica serovar Typhimurium as a model of human systemic invasive infection. In this model, strain CC042/GeniUnc (CC042) mice displayed extreme susceptibility with very high bacterial loads and mortality. CC042 mice showed lower spleen weights and decreased splenocyte numbers before and after infection, affecting mostly CD8+ T cells, B cells, and all myeloid cell populations, compared with control C57BL/6J mice. CC042 mice also had lower thymus weights with a reduced total number of thymocytes and double-negative and double-positive (CD4+, CD8+) thymocytes compared to C57BL/6J mice. Analysis of bone marrow-resident hematopoietic progenitors showed a strong bias against lymphoid-primed multipotent progenitors. An F2 cross between CC042 and C57BL/6N mice identified two loci on chromosome 7 (Stsl6 and Stsl7) associated with differences in bacterial loads. In the Stsl7 region, CC042 carried a loss-of-function variant, unique to this strain, in the integrin alpha L (Itgal) gene, the causative role of which was confirmed by a quantitative complementation test. Notably, Itgal loss of function increased the susceptibility to S. Typhimurium in a (C57BL/6J × CC042)F1 mouse background but not in a C57BL/6J mouse inbred background. These results further emphasize the utility of the Collaborative Cross to identify new host genetic variants controlling susceptibility to infections and improve our understanding of the function of the Itgal gene

Functions of lipid raft membrane microdomains at the blood-brain barrier

The blood-brain barrier (BBB) is a highly specialized structural and functional component of the central nervous system that separates the circulating blood from the brain and spinal cord parenchyma. Brain endothelial cells (BECs) that primarily constitute the BBB are tightly interconnected by multiprotein complexes, the adherens junctions and the tight junctions, thereby creating a highly restrictive cellular barrier. Lipid-enriched membrane microdomain compartmentalization is an inherent property of BECs and allows for the apicobasal polarity of brain endothelium, temporal and spatial coordination of cell signaling events, and actin remodeling. In this manuscript, we review the role of membrane microdomains, in particular lipid rafts, in the BBB under physiological conditions and during leukocyte transmigration/diapedesis. Furthermore, we propose a classification of endothelial membrane microdomains based on their function, or at least on the function ascribed to the molecules included in such heterogeneous rafts: (1) rafts associated with interendothelial junctions and adhesion of BECs to basal lamina (scaffolding rafts); (2) rafts involved in immune cell adhesion and migration across brain endothelium (adhesion rafts); (3) rafts associated with transendothelial transport of nutrients and ions (transporter rafts)