ALCAM : cell adhesion molecule or tight junction? The characterization of its role in the context of neuroinflammation

But : La perte de l’intégrité de la barrière hémo-encéphalique (BHE) est l’une des caractéristiques principales de la sclérose en plaques. Cette augmentation de la perméabilité est associée à une désorganisation des molécules de jonction serrée et à une augmentation de l’expression de molécules d’adhérence essentielles à l’extravasation des cellules immunitaires. Identifier de nouvelles molécules impliquées dans ce processus est donc crucial pour le développement de nouvelles thérapies contre la sclérose en plaques visant à promouvoir l’intégrité de la BHE et à diminuer la migration des leucocytes dans le système nerveux central (SNC) au cours du processus neuro-inflammatoire. Dans cette étude, le rôle spécifique de la molécule d’adhérence ALCAM, qui est exprimé à la surface des cellules endothéliales de la BHE (CE-BHE) et de certains sous-types de leucocytes, a été évalué. Méthodologie : À l’aide d’une analyse protéomique exhaustive, notre laboratoire a identifié ALCAM comme étant une molécule d’adhérence surexprimée par les CE-BHE mises en culture dans un milieu pro-inflammatoire. Dans le but d’étudier le rôle spécifique d’ALCAM durant la diapédèse leucocytaire, nous avons induit chez des souris de type sauvages et des souris ALCAM déficientes l’encéphalite auto-immune expérimentale (EAE), le modèle animal de la sclérose en plaques. Le rôle d’ALCAM a aussi été étudié à l’aide d’un système d’adhérence sous flux laminaire. Cet appareil, qui imite un capillaire cérébral, permet de suivre en temps réel le mouvement des leucocytes, soumis à une pression physiologique, dans un tube couvert à sa base par des CE-BHE. Résultats : En utilisant ce système d’adhérence, j’ai pu démontrer que des anticorps dirigés contre ALCAM réduisent de façon significative le roulement et l’adhérence de monocytes CD14+ humains à la surface de CE-BHE. Par ailleurs, ces anticorps préviennent de façon marquée la diminution de la vitesse moyenne des cellules au cours de l’expérience. Par le fait même, j’ai aussi observé une réduction significative de l’extravasation des monocytes traités avec de l’anti-ALCAM au travers de CE-BHE dans un modèle statique de migration. Subséquemment, j’ai démontré que ces monocytes migrent plus rapidement et en plus grand nombre au travers d’une barrière constituée de cellules endothéliales méningées à comparer à des CE-BHE. Bien que des observations similaires ont été effectuées en utilisant des lymphocytes T CD4+ humains ex vivo, j’ai été incapable de reproduire ces résultats à l’aide de cellules Th1 et Th17 réactivées in vitro. Par opposition à nos données in vitro, j’ai découvert que les souris déficientes en ALCAM développent une EAE active plus sévère que celle observée chez des souris de type sauvages. Cette EAE est par ailleurs associée à une infiltration périvasculaire de lymphocytes T pro-inflammatoires et de monocytes/macrophages de type M1 plus marqué chez les souris ALCAM déficientes. L’induction d’une EAE par transfert adoptif, dans laquelle des cellules immunitaires de type sauvage réactivées par du MOG sont injectées à des souris déficientes en ALCAM, suggère que la pathophysiologie observée durant l’EAE active serait liée à l’absence d’ALCAM au niveau de la BHE. Une caractérisation de la barrière des souris ALCAM déficientes non immunisées a par la suite révélé une réduction de l’expression de certaines molécules de jonction serrée. Une analyse plus poussée a par ailleurs démontré qu’ALCAM est lié indirectement à des molécules de jonction serrée des CE-BHE, ce qui expliquerait l’augmentation de la perméabilité de celle-ci chez les souris déficientes en ALCAM. Une analyse de la perméabilité intercellulaire de la BHE effectuée in vitro a d’autre part corrélé ces résultats. Conclusion : Collectivement, nos données prouvent qu’ALCAM joue un rôle prépondérant dans la diapédèse des monocytes, mais pas des lymphocytes Th1 et Th17 au travers de la BHE. Par ailleurs, nos résultats suggèrent qu’ALCAM remplit une fonction biologique cruciale favorisant le maintien de l’intégrité de la BHE en agissant comme molécule adaptatrice intermédiaire entre les molécules de jonction serrées et le cytosquelette. De cette façon, l’absence d’ALCAM au niveau des CE-BHE promeut indirectement le recrutement de leucocytes pro-inflammatoires dans le SNC des souris atteintes de l’EAE en augmentant la perméabilité des vaisseaux sanguins de la BHE.Aim: The loss of blood-brain barrier (BBB) integrity is a hallmark of multiple sclerosis. It is associated with a disorganization of junctional molecules and an upregulation of cell adhesion molecules essential for immune cell transmigration. Identifying novel key players involved in this process is thus crucial for the development of MS therapies aimed at promoting BBB integrity and decreasing leukocytes trafficking into the central nervous system (CNS) during neuroinflammation. In this study, the specific role of the adhesion molecule ALCAM, found on BBB endothelial cells (BBB-ECs) and subsets of leukocytes, was assessed. Methods: We first identified ALCAM as an important molecule upregulated during inflammation in a proteomic screen of in vitro cultured primary human BBB-ECs. In order to study the effects of ALCAM on leukocyte transmigration, both active and passive experimental autoimmune encephalomyelitis (EAE) was induced in ALCAM KO and WT animals. The specific role of ALCAM during leukocyte transmigration was also assessed using a modified adhesion assay under sheer-stress, in which leukocytes flow across a capillary-like channel lined with a monolayer of BBB-ECs under physiological pressure. Results: Using the modified adhesion assay, we demonstrated that anti-ALCAM blocking antibodies significantly reduce the rolling and the adhesion of human CD14+ monocytes interacting with primary human BBB-ECs, as well as prevent their overall decrease in velocity. Concurrently, we also observed a significant reduction in the migration of ex vivo CD14+ monocytes, across a monolayer of human BBB-ECs. These monocytes also migrated more rapidly and in higher number across meningeal endothelial cells, as compared to BBB-ECs. While similar observations were made using ex vivo CD4+ T lymphocytes, we failed to reproduce these results using in vitro activated Th1 and Th17 cells. In opposition to our in vitro data, ALCAM KO mice developed a more severe active EAE associated with a significant increase in perivascular infiltration of pro-inflammatory lymphocytes (Th1/Th17) and M1 monocytes/macrophages, as compared to WT controls. In addition, EAE transfer experiments, in which ALCAM KO mice received WT MOG-reactivated splenocytes, suggested that the pathophysiology observed in active EAE was linked to the absence of ALCAM on BBB-ECs. Phenotypic characterization of un-immunized ALCAM KO mice revealed a reduced expression of BBB junctional proteins. Further analysis showed that ALCAM is indirectly associated with tight junction molecules of the BBB-ECs, which explains the increased CNS parenchymal blood vessel in vivo permeability in ALCAM KO animals. Correlating with these data, primary culture of mouse brain BBB-ECs was shown to possess a lower TEER and an increased permeability coefficient. Conclusion: Collectively, our data provide evidence of the implication of ALCAM in monocyte transmigration, but not Th1 or Th17 lymphocyte diapedesis across CNS endothelium. Our results also point to a biologically crucial function of ALCAM in maintaining BBB integrity by acting as an adaptor molecule between tight junctions and the cytoskeleton. As such, the absence of ALCAM at the level of BBB-ECs indirectly promotes the recruitment of pro-inflammatory leukocytes in the CNS of EAE animals by increasing the BBB vessels permeability

Isolation of endothelial cells, pericytes and astrocytes from mouse brain.

Primary cell isolation from the central nervous system (CNS) has allowed fundamental understanding of blood-brain barrier (BBB) properties. However, poorly described isolation techniques or suboptimal cellular purity has been a weak point of some published scientific articles. Here, we describe in detail how to isolate and enrich, using a common approach, endothelial cells (ECs) from adult mouse brains, as well as pericytes (PCs) and astrocytes (ACs) from newborn mouse brains. Our approach allowed the isolation of these three brain cell types with purities of around 90%. Furthermore, using our protocols, around 3 times more PCs and 2 times more ACs could be grown in culture, as compared to previously published protocols. The cells were identified and characterized using flow cytometry and confocal microscopy. The ability of ECs to form a tight monolayer was assessed for passages 0 to 3. The expression of claudin-5, occludin, zonula occludens-1, P-glycoprotein-1 and breast cancer resistance protein by ECs, as well as the ability of the cells to respond to cytokine stimuli (TNF-α, IFN-γ) was also investigated by q-PCR. The transcellular permeability of ECs was evaluated in the presence of pericytes or astrocytes in a Transwell® model by measuring the transendothelial electrical resistance (TEER), dextran-FITC and sodium fluorescein permeability. Overall, ECs at passages 0 and 1 featured the best properties valued in a BBB model. Furthermore, pericytes did not increase tightness of EC monolayers, whereas astrocytes did regardless of their seeding location. Finally, ECs resuspended in fetal bovine serum (FBS) and dimethyl sulfoxide (DMSO) could be cryopreserved in liquid nitrogen without affecting their phenotype nor their capacity to form a tight monolayer, thus allowing these primary cells to be used for various longitudinal in vitro studies of the blood-brain barrier

Dual role of ALCAM in neuroinflammation and blood-brain barrier homeostasis.

Activated leukocyte cell adhesion molecule (ALCAM) is a cell adhesion molecule found on blood-brain barrier endothelial cells (BBB-ECs) that was previously shown to be involved in leukocyte transmigration across the endothelium. In the present study, we found that ALCAM knockout (KO) mice developed a more severe myelin oligodendrocyte glycoprotein (MOG)35-55-induced experimental autoimmune encephalomyelitis (EAE). The exacerbated disease was associated with a significant increase in the number of CNS-infiltrating proinflammatory leukocytes compared with WT controls. Passive EAE transfer experiments suggested that the pathophysiology observed in active EAE was linked to the absence of ALCAM on BBB-ECs. In addition, phenotypic characterization of unimmunized ALCAM KO mice revealed a reduced expression of BBB junctional proteins. Further in vivo, in vitro, and molecular analysis confirmed that ALCAM is associated with tight junction molecule assembly at the BBB, explaining the increased permeability of CNS blood vessels in ALCAM KO animals. Collectively, our data point to a biologically important function of ALCAM in maintaining BBB integrity

Neutrophils mediate blood-spinal cord barrier disruption in demyelinating neuroinflammatory diseases

Disruption of the blood-brain and blood-spinal cord barriers (BBB and BSCB, respectively) and immune cell infiltration are early pathophysiological hallmarks of multiple sclerosis (MS), its animal model experimental autoimmune encephalomyelitis (EAE), and neuromyelitis optica (NMO). However, their contribution to disease initiation and development remains unclear. In this study, we induced EAE in lys-eGFP-ki mice and performed single, nonterminal intravital imaging to investigate BSCB permeability simultaneously with the kinetics of GFP(+) myeloid cell infiltration. We observed a loss in BSCB integrity within a day of disease onset, which paralleled the infiltration of GFP(+) cells into the CNS and lasted for ∼4 d. Neutrophils accounted for a significant proportion of the circulating and CNS-infiltrating myeloid cells during the preclinical phase of EAE, and their depletion delayed the onset and reduced the severity of EAE while maintaining BSCB integrity. We also show that neutrophils collected from the blood or bone marrow of EAE mice transmigrate more efficiently than do neutrophils of naive animals in a BBB cell culture model. Moreover, using intravital videomicroscopy, we demonstrate that the IL-1R type 1 governs the firm adhesion of neutrophils to the inflamed spinal cord vasculature. Finally, immunostaining of postmortem CNS material obtained from an acutely ill multiple sclerosis patient and two neuromyelitis optica patients revealed instances of infiltrated neutrophils associated with regions of BBB or BSCB leakage. Taken together, our data provide evidence that neutrophils are involved in the initial events that take place during EAE and that they are intimately linked with the status of the BBB/BSCB

MicroRNA-223 protects neurons from degeneration in experimental autoimmune encephalomyelitis

Multiple sclerosis is a chronic inflammatory, demyelinating, and neurodegenerative disease affecting the brain, spinal cord and optic nerves. Neuronal damage is triggered by various harmful factors that engage diverse signalling cascades in neurons; thus, therapeutic approaches to protect neurons will need to focus on agents that can target multiple biological processes. We have therefore focused our attention on microRNAs: small non-coding RNAs that primarily function as post-transcriptional regulators that target messenger RNAs and repress their translation into proteins. A single microRNA can target many functionally related messenger RNAs making microRNAs powerful epigenetic regulators. Dysregulation of microRNAs has been described in many neurodegenerative diseases including multiple sclerosis. Here, we report that two microRNAs, miR-223-3p and miR-27a-3p, are upregulated in neurons in the experimental autoimmune encephalomyelitis mouse model of CNS inflammation and in grey matter-containing multiple sclerosis lesions. Prior work has shown peripheral blood mononuclear cell conditioned media causes sublethal degeneration of neurons in culture. We find overexpression of miR-27a-3p or miR-223-3p protects dissociated cortical neurons from condition media mediated degeneration. Introduction of miR-223-3p in vivo in mouse retinal ganglion cells protects their axons from degeneration in experimental autoimmune encephalomyelitis. In silico analysis revealed that messenger RNAs involved in glutamate receptor signalling are enriched as miR-27a-3p and miR-223-3p targets. We observe that antagonism of NMDA and AMPA type glutamate receptors protects neurons from condition media dependent degeneration. Our results suggest that miR-223-3p and miR-27a-3p are upregulated in response to inflammation to mediate a compensatory neuroprotective gene expression program that desensitizes neurons to glutamate by targeting messenger RNAs involved in glutamate receptor signalling

Neutrophils Mediate Blood–Spinal Cord Barrier Disruption in Demyelinating Neuroinflammatory Diseases

Disruption of the blood-brain and blood-spinal cord barriers (BBB and BSCB, respectively) and immune cell infiltration are early pathophysiological hallmarks of multiple sclerosis (MS), its animal model experimental autoimmune encephalomyelitis (EAE), and neuromyelitis optica (NMO). However, their contribution to disease initiation and development remains unclear. In this study, we induced EAE in lys-eGFP-ki mice and performed single, nonterminal intravital imaging to investigate BSCB permeability simultaneously with the kinetics of GFP(+) myeloid cell infiltration. We observed a loss in BSCB integrity within a day of disease onset, which paralleled the infiltration of GFP(+) cells into the CNS and lasted for ∼4 d. Neutrophils accounted for a significant proportion of the circulating and CNS-infiltrating myeloid cells during the preclinical phase of EAE, and their depletion delayed the onset and reduced the severity of EAE while maintaining BSCB integrity. We also show that neutrophils collected from the blood or bone marrow of EAE mice transmigrate more efficiently than do neutrophils of naive animals in a BBB cell culture model. Moreover, using intravital videomicroscopy, we demonstrate that the IL-1R type 1 governs the firm adhesion of neutrophils to the inflamed spinal cord vasculature. Finally, immunostaining of postmortem CNS material obtained from an acutely ill multiple sclerosis patient and two neuromyelitis optica patients revealed instances of infiltrated neutrophils associated with regions of BBB or BSCB leakage. Taken together, our data provide evidence that neutrophils are involved in the initial events that take place during EAE and that they are intimately linked with the status of the BBB/BSCB