13 research outputs found
Human brain endothelial cells endeavor to immunoregulate CD8 T cells via PD-1 ligand expression in multiple sclerosis
Background
Multiple sclerosis (MS), an inflammatory disease of the central nervous system (CNS), is characterized by blood-brain barrier (BBB) disruption and massive infiltration of activated immune cells. Engagement of programmed cell death-1 (PD-1) expressed on activated T cells with its ligands (PD-L1 and PD-L2) suppresses T cell responses. We recently demonstrated in MS lesions elevated PD-L1 expression by glial cells and absence of PD-1 on many infiltrating CD8 T cells. We have now investigated whether human brain endothelial cells (HBECs), which maintain the BBB, can express PD-L1 or PD-L2 and thereby modulate T cells.
Methods
We used primary cultures of HBECs isolated from non-tumoral CNS tissue either under basal or inflamed conditions. We assessed the expression of PD-L1 and PD-L2 using qPCR and flow cytometry. Human CD8 T cells were isolated from peripheral blood of healthy donors and co-cultured with HBECs. Following co-culture with HBECs, proliferation and cytokine production by human CD8 T cells were measured by flow cytometry whereas transmigration was determined using a well established in vitro model of the BBB. The functional impact of PD-L1 and PD-L2 provided by HBECs was determined using blocking antibodies. We performed immunohistochemistry for the detection of PD-L1 or PD-L2 concurrently with caveolin-1 (a cell specific marker for endothelial cells) on post-mortem human brain tissues obtained from MS patients and normal controls.
Results
Under basal culture conditions, PD-L2 is expressed on HBECs, whilst PD-L1 is not detected. Both ligands are up-regulated under inflammatory conditions. Blocking PD-L1 and PD-L2 leads to increased transmigration and enhanced responses by human CD8 T cells in co-culture assays. Similarly, PD-L1 and PD-L2 blockade significantly increases CD4 T cell transmigration. Brain endothelium in normal tissues and MS lesions does not express detectable PD-L1; in contrast, all blood vessels in normal brain tissues are PD-L2-positive, while only about 50% express PD-L2 in MS lesions.
Conclusions
Our observations suggest that brain endothelial cells contribute to control T cell transmigration into the CNS and immune responses via PD-L2 expression. However, such impact is impaired in MS lesions due to downregulation of endothelium PD-L2 levels
Expression et rÎle de PD-1 et de ses ligands dans le contexte de la sclérose en plaques
La sclĂ©rose en plaques (SEP) est une maladie inflammatoire dĂ©myĂ©linisante et neurodĂ©gĂ©nĂ©rative du systĂšme nerveux central (SNC). Les cellules T activĂ©es qui expriment le PD-1 sont inhibĂ©es via lâinteraction avec lâun des ligands: PD-L1 ou PD-L2. Des Ă©tudes effectuĂ©es chez le modĂšle murin de la SEP, lâencĂ©phalomyĂ©lite auto-immune expĂ©rimentale (EAE), ont dĂ©montrĂ© que lâinteraction du PD-1 avec ses ligands contribue Ă attĂ©nuer la maladie. Toutefois, le rĂŽle du PD-1 et de ses ligands dans la pathogenĂšse de la SEP chez lâhumain et dans le modĂšle murin nâa pas Ă©tĂ© complĂštement Ă©lucidĂ©.
Nous avons dĂ©terminĂ© que plusieurs cellules du SNC humain peuvent exprimer les ligands du PD-1. Les astrocytes, les microglies, les oligodendrocytes et les neurones expriment faiblement le PD-L1 dans des conditions basales mais augmentent de façon significative cette expression en rĂ©ponse Ă des cytokines inflammatoires. Le blocage de lâexpression du PD-L1 par les astrocytes Ă lâaide de siRNA spĂ©cifiques mĂšne Ă lâaugmentation significative des rĂ©ponses des cellules T CD8+ (prolifĂ©ration, cytokines, enzymes lytiques). Nos rĂ©sultats Ă©tablissent ainsi que les cellules gliales humaines peuvent exprimer des niveaux suffisants de PD-L1 en milieu inflammatoire pour inhiber les rĂ©ponses des cellules T CD8+. Notre analyse de tissus cĂ©rĂ©braux post-mortem par immunohistochimie dĂ©montre que dans les lĂ©sions de la SEP les niveaux de PD-L1 sont significativement plus Ă©levĂ©s que dans les tissus de tĂ©moins; les astrocytes et les microglies/macrophages expriment le PD-L1. Cependant, plus de la moitiĂ© des lymphocytes T CD8+ ayant infiltrĂ© des lĂ©sions de SEP nâexpriment pas le rĂ©cepteur PD-1. Au cours du dĂ©veloppement de lâEAE, les cellules du SNC augmentent leur niveau de PD-L1. Le PD-1 est fortement exprimĂ© par les cellules T dĂšs le dĂ©but des symptĂŽmes, mais son intensitĂ© diminue au cours de la maladie, rendant les cellules T insensibles au signal inhibiteur envoyĂ© par le PD-L1.
Nous avons observĂ© que les cellules endothĂ©liales humaines formant la barriĂšre hĂ©mato-encĂ©phalique (BHE) expriment de façon constitutive le PD-L2 mais pas le PD-L1 et que lâexpression des deux ligands augmente dans des conditions inflammatoires. Les ligands PD-L1 et PD-L2 exprimĂ©s par les cellules endothĂ©liales ont la capacitĂ© de freiner lâactivation des cellules T CD8+ et CD4+, ainsi que leur migration Ă travers la BHE. LâendothĂ©lium du cerveau des tissus normaux et des lĂ©sions SEP nâexprime pas des taux dĂ©tectables de PD-L1. En revanche, tous les vaisseaux sanguins des tissus de cerveaux normaux sont positifs pour le PD-L2, alors que seulement la moitiĂ© de ceux-ci expriment le PD-L2 dans des lĂ©sions SEP.
Nos travaux dĂ©montrent que lâentrĂ©e des cellules T activĂ©es est contrĂŽlĂ©e dans des conditions physiologiques grĂące Ă la prĂ©sence du PD-L2 sur la BHE. Cependant, lâexpression plus faible du PD-L2 sur une partie des vaisseaux sanguins dans les lĂ©sions SEP nuit au contrĂŽle de la migration des cellules immunes. De plus, une fois dans le SNC, les cellules T CD8+ Ă©tant dĂ©pourvues du PD-1 ne peuvent recevoir le signal inhibiteur fourni par le PD-L1 fortement exprimĂ© par les cellules du SNC, leur permettant ainsi de rester activĂ©es.Multiple sclerosis (MS) is an inflammatory, demyelinating and neurodegenerative disease of the central nervous system (CNS). Responses of activated T cells are suppressed upon engagement of the receptor programmed cell death-1 (PD-1) with its ligands (PD-L1 and PD-L2). Experiments using the mouse model of MS, experimental autoimmune encephalomyelitis (EAE), have demonstrated that the PD-1/PD-Ls interaction contributes to attenuate disease severity. However, the expression and the role of PD-1 and PD-Ls have been partially documented in inflammatory murine models and human CNS data are still incomplete.
We determined that primary cultures of human astrocytes, microglia, oligodendrocytes, or neurons expressed low or undetectable PD-L1 levels under basal conditions, but inflammatory cytokines significantly induced such expression, especially on astrocytes and microglia. Blocking PD-L1 expression in astrocytes using specific siRNA in co-culture led to significantly increased CD8 T cell responses (proliferation, cytokines, lytic enzyme). Thus, our results establish that inflamed human glial cells can express sufficient and functional PD-L1 to inhibit CD8 T cell responses. Extensive immunohistochemical analysis of post-mortem brain tissues demonstrated a significantly greater PD-L1 expression in MS lesions compared to control tissues, which co-localized with astrocyte and microglia/macrophage cell markers. However, more than half of infiltrating CD8 T lymphocytes in MS lesions did not express PD-1, the cognate receptor. Similar results were obtained in EAE mice. Even though CNS cells expressed PD-L1 at the peak of the disease, PD-1 intensity on infiltrating T cells decreased throughout EAE disease development. This reduction of PD-1 level on activated T cells prevented these cells to receive PD-L1 inhibitory signal.
We also investigated whether human brain endothelial cells (HBECs), which form the blood brain barrier (BBB), can express PD-L1 or PD-L2 and thereby modulate T cells. HBECs expressed PD-L2 under basal conditions, whilst PD-L1 was not detected. Both ligands were up-regulated under inflammatory conditions. Blocking PD-L1 and PD-L2 led to increased transmigration and enhanced responses by human CD8 T cells in co-culture assays. Similarly, PD-L1 and PD-L2 blockade significantly increased CD4 T cell transmigration. Brain endothelium in normal tissues and MS lesions did not express detectable PD-L1; in contrast, all blood vessels in normal brain tissues were PD-L2-positive, while only about 50% expressed PD-L2 in MS lesions.
Therefore, our results demonstrate that under basal conditions, PD-L2 expression by HBECs impedes the migration of activated immune T cells through the BBB, and inhibits their activation. However, such impact is impaired in MS lesions due to down-regulation of PD-L2 levels on the endothelium. The majority of infiltrating CD8 T cells is devoid of PD-1, thus insensitive to PD-L1 inhibitory signal providing by CNS cells once they have entered the CNS
Le consentement du lésé en droit pénal suisse: Conditions et validité dans le domaine médical
Cette contribution se penche sur la place et la portĂ©e du consentement du lĂ©sĂ© en droit pĂ©nal suisse, et plus particuliĂšrement dans le domaine mĂ©dical. Les autrices prĂ©sentent tout dâabord le champ dâapplication de ce fait justificatif â qui se dĂ©termine en tenant compte, notamment, du bien juridiquement protĂ©gĂ© par la disposition pĂ©nale concernĂ©e. Les autrices discutent de la place que le consentement du lĂ©sĂ© peut prendre au regard de la thĂ©orie de lâinfraction. Elles prĂ©sentent ensuite le contexte dans lequel le consentement est donnĂ© et la lĂ©sion survient, avant dâen analyser la portĂ©e en droit mĂ©dical. Finalement, les autrices proposent une liste des conditions dâapplication du consentement du lĂ©sĂ© en portant une attention particuliĂšre aux Ă©lĂ©ments spĂ©cifiques et problĂ©matiques du domaine mĂ©dical
Contribution of Astrocyte-Derived IL-15 to CD8 T Cell Effector Functions in Multiple Sclerosis
The Role of Prophage for Genome Diversification within a Clonal Lineage of Lactobacillus johnsonii: Characterization of the Defective Prophage LJ771âż â
Two independent isolates of the gut commensal Lactobacillus johnsonii were sequenced. These isolates belonged to the same clonal lineage and differed mainly by a 40.8-kb prophage, LJ771, belonging to the Sfi11 phage lineage. LJ771 shares close DNA sequence identity with Lactobacillus gasseri prophages. LJ771 coexists as an integrated prophage and excised circular phage DNA, but phage DNA packaged into extracellular phage particles was not detected. Between the phage lysin gene and attR a likely mazE (âantitoxinâ)/pemK (âtoxinâ) gene cassette was detected in LJ771 but not in the L. gasseri prophages. Expressed pemK could be cloned in Escherichia coli only together with the mazE gene. LJ771 was shown to be highly stable and could be cured only by coexpression of mazE from a plasmid. The prophage was integrated into the methionine sulfoxide reductase gene (msrA) and complemented the 5âČ end of this gene, creating a protein with a slightly altered N-terminal sequence. The two L. johnsonii strains had identical in vitro growth and in vivo gut persistence phenotypes. Also, in an isogenic background, the presence of the prophage resulted in no growth disadvantage
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