Ninjurin-1 est une molécule d'adhérence de la barrière hémato-encéphalique impliquée dans le recrutement de monocytes au sein du système nerveux central

La sclérose en plaques (SEP) est caractérisée par des infiltrations périvasculaires de cellules immunitaires et par de la démyélinisation au sein du système nerveux central (SNC). Ces deux paramètres de la maladie sont associés à la fragilisation de la barrière hémato-encéphalique (BHE). En ce sens, le recrutement des cellules présentatrices d’antigène (CPA) myéloïdes, telles que les monocytes, les macrophages et les cellules dendritiques, dans le SNC à travers la BHE, est une étape cruciale dans l’initiation et la persistance de l’inflammation cérébrale. Nerve injury-induced protein (Ninjurin)-1 est une nouvelle molécule d’adhérence qui médie une interaction de type homophilique et dont l’expression sur l’endothélium vasculaire de la BHE humaine fut identifiée grâce à une analyse protéomique des protéines associées à la BHE. Les résultats présentés dans ce mémoire montrent que l’expression de Ninjurin-1 augmente dans un contexte inflammatoire dans les cultures primaires de cellules endothéliales de la BHE (CE-BHE) et sur les CPA myéloïdes humaines ex vivo et générées in vitro. De plus, les CPA infiltrantes retrouvées dans les lésions cérébrales de patients atteints de SEP et dans le SNC des souris atteintes d’encéphalomyélite autoimmune expérimentale (EAE), le modèle murin de la SEP, expriment de hauts niveaux de Ninjurin-1. À l’aide du modèle in vitro de la BHE, la neutralisation de Ninjurin-1 restreint spécifiquement la migration des monocytes à travers les CE-BHE sans affecter le recrutement des lymphocytes, ni la perméabilité des CE-BHE. Enfin, les souris atteintes d’EAE et traitées avec un peptide bloquant dirigé contre Ninjurin-1 présentent une maladie moins sévère ainsi qu’une diminution des CPA infiltrant le SNC et ce comparé au groupe contrôle. Ces résultats suggèrent que Ninjurin-1 est une molécule d’adhérence de la BHE impliquée dans le recrutement de CPA myéloïdes au sein du SNC et qu’elle peut être considérée comme une cible thérapeutique potentielle en SEP.Multiple Sclerosis (MS) is characterized by perivascular infiltrations of immune cells and by demyelination in the central nervous system (CNS). These two hallmarks of the disease are associated with the disruption of the blood-brain barrier (BBB). The recruitment of monocytes, macrophages and dendritic cells, the so-called myeloid antigen-presenting cells (APCs), in the CNS through the BBB is thought to play a crucial role in the initiation and the persistence of the disease. Therefore the identification of the molecular mechanisms involved in the migration of myeloid APCs into the CNS is considered a valid therapeutic option in MS. Nerve injury-induced protein (Ninjurin)-1, a novel adhesion molecule that mediates homophilic binding, was found to be expressed in the vascular endothelium of the BBB following a proteomic screen of human BBB-associated proteins. Ninjurin-1’s expression increases during an inflammatory context in primary cultures of endothelial cells of the BBB (BBB-ECs) and on ex vivo and in vitro generated myeloid APCs. In addition, infiltrating APCs in human MS lesions and in the CNS of the murine model of MS, the mice affected with experimental autoimmune encephalomyelitis (EAE), express high levels of Ninjurin-1. Using an experimental model of the BBB, the neutralization of Ninjurin-1 specifically restricts the migration of monocytes across the BBB-ECs without affecting the recruitment of lymphocytes or the permeability of the BBB-ECs. Finally, EAE mice treated with a Ninjurin-1 blocking peptide have reduced disease severity and a reduced infiltration of myeloid APCs in the CNS, as compared to the control group. Our results show that Ninjurin-1 is an adhesion molecule of the BBB involved in the recruitment of myeloid APCs to the CNS and is also a potential therapeutic target to dampen CNS inflammatory processes, as occurs in MS

OR.21. MCAM/CD146 is Expressed by Brain Endothelial Cells and Defines a Unique Effector Memory Lymphocyte Subset Involved in Neuroinflammation

Immune cell infiltration to the central nervous system (CNS) is a hallmark of the disease Multiple Sclerosis (MS). In MS, BBB dysfunction is associated with increased expression of adhesion molecules and chemokines leading to increased leukocyte trafficking to the CNS. The reciprocal attachment of leukocytes to BBB endothelial cells (BBB-ECs) is a key step of this CNS infiltration. Cell adhesion molecules (CAM) of the of the immunoglobulin family (ICAM-1 and VCAM) are key players in the interactions between the immune cells and the BBB-ECs. To describe novel CAMs of the BBB we used a lipid raft-based proteomic approach to identify an immunoglobulin subfamily of proteins expressed by BBB-ECs. Melanoma cell adhesion molecule (MCAM/CD146) was identified in this screen and this project aims to characterize MCAM's function in immune cell migration across the BBB. Our data demonstrates that MCAM is expressed on the surface of BBB-ECs, in vitro and in situ and co-localizes with lymphocyte surface markers during adhesion and diapedesis. Since MCAM's only known ligand is MCAM itself, we analysed MCAM expression on peripheral blood leukocytes from human healthy donors and MS patients. MCAM was detected on a unique subset of effector memory CD4+and CD8+T lymphocytes. In vitro polyclonal activation induced MCAM up-regulation on T lymphocytes. Moreover, we found that MCAM+T lymphocytes produce significantly more inflammatory cytokines interferon-gamma and IL-17 compared to MCAMneg cells. Finally, the proportion of MCAM+T cells was found to be consistently higher in MS patients during relapses compared to non-relapsing patients and controls. Our data indicates that MCAM is expressed by both the BBB and peripheral blood immune cells and suggests that MCAM could play an important role in CNS inflammatory reactions, including MS.Peer reviewed: YesNRC publication: Ye

Endo-MitoEGFP mice: a novel transgenic mouse with fluorescently marked mitochondria in microvascular endothelial cells.

Blood vessel-specific fluorescent transgenic mice are excellent tools to study the development of the vasculature and angiogenic processes. There is growing interest in the biological processes relevant to endothelial cells but limited tools exist to selectively evaluate subcellular functions of this cell type in vivo. Here, we report a novel transgenic animal model that expresses mitochondrially targeted enhanced green fluorescent protein (EGFP) via the Hb9 promoter, a homeobox transcription factor with limited known involvement in the vasculature. Random integration of the transgene, containing the entire mouse Hb9 promoter, was found to be expressed in a variety of vascularised tissues. Further inspection revealed that Mito-EGFP localizes to the endothelial cells (ECs) of a subset of microvascular blood vessels, especially in the central nervous system (CNS), heart, spleen, thymus, lymph nodes and skin. We demonstrate the utility of this novel transgenic mouse, named Endo-MitoEGFP, in the detection, imaging, and isolation of microvascular ECs and evaluation of EC mitochondrial function isolated from adult animals. These transgenic mice will be useful to studies of ECs in development, physiology, and pathology

Role of Ninjurin-1 in the migration of myeloid cells to central nervous system inflammatory lesions

Peer reviewed: YesNRC publication: Ye

EGFP expression is detected in ECs via flow cytometry.

<p>(A) Representative dot plot of ECs isolated from spleens of transgenic mice. The EC population was selected based on size and labelling with PECAM-1. (B) The majority of splenic ECs express EGFP and PECAM-1. (C) Western blot of brains homogenates (H), as well as soluble (S1) and insoluble (S2) fractions from lysates of ECs isolated from CNS vessels of transgenic animals and immunoblotted for EGFP and p120. <i>n</i>=3 animals.</p

EGFP expression is not restricted to central nervous system, but is also expressed in vascularized tissues.

<p>(A) Schematic of p<i>Hb9</i>-MitoEGFP transgenic construct, with mitochondrial targeting sequence of Cytochrome <i>c</i> Oxidase subunit VIII. (B) RT-PCR of EGFP mRNA from a panel of tissues of transgenic (+) and non-transgenic littermates (-). Actin serves as a loading control. (C) EGFP protein levels detected via immunoblotting in a panel of tissues isolated from transgenic (+) and non-transgenic littermates (-). SOD1 serves as a loading control. <i>n</i>=3-4 animals.</p

EGFP is expressed within mitochondria.

<p>(A) Homogenates (H), cytosolic protein (C) and purified mitochondria (M) were collected from the brain, spinal cord and spleen of transgenic mice and probed for EGFP via immunoblot. SOD1 and VDAC serve as controls for cytosol and mitochondria, respectively. (B) Mitochondria were isolated from the spleens of transgenic and non-transgenic mice. <i>Left</i>: Mitochondria were gated according to their light scattering properties (forward scatter, FSC; side scatter, SSC). <i>Middle</i>: Gated mitochondria were stained with MitoTracker Red (MTR, black, dashed) and compared to unstained (grey, shaded) mitochondria. <i>Right</i>: MTR⁺ mitochondria from transgenic (black, unshaded) and non-transgenic (grey, shaded) mice were analyzed for EGFP expression. Data presented is representative of three independent experiments. (C) Mitochondria from transgenic (black, unshaded), and non-transgenic (grey, shaded) mice were analyzed for EGFP expression. (D) The transmembrane potential of EGFP⁺ mitochondria was assayed using TMRM. Mitochondria were left, unstained (grey, filled), treated with TMRM, basal conditions (blue), or treated with the protonophore CCCP (red). (E) Mitochondrial superoxide production of EGFP⁺ mitochondria, was assayed using MitoSOX Red. Mitochondria were left, unstained (grey, filled), treated with MitoSOX Red under basal conditions (green), or treated with the complex III inhibitor Antimycin A (orange). C, D, E are from the same sample and are representative of three independent experiments.</p