RESUMEN
The trafficking of autoreactive leucocytes across the blood-brain barrier endothelium is a hallmark of multiple sclerosis pathogenesis. Although the blood-brain barrier endothelium represents one of the main CNS borders to interact with the infiltrating leucocytes, its exact contribution to neuroinflammation remains understudied. Here, we show that Mcam identifies inflammatory brain endothelial cells with pro-migratory transcriptomic signature during experimental autoimmune encephalomyelitis. In addition, MCAM was preferentially upregulated on blood-brain barrier endothelial cells in multiple sclerosis lesions in situ and at experimental autoimmune encephalomyelitis disease onset by molecular MRI. In vitro and in vivo, we demonstrate that MCAM on blood-brain barrier endothelial cells contributes to experimental autoimmune encephalomyelitis development by promoting the cellular trafficking of TH1 and TH17 lymphocytes across the blood-brain barrier. Last, we showcase ST14 as an immune ligand to brain endothelial MCAM, enriched on CD4+ T lymphocytes that cross the blood-brain barrier in vitro, in vivo and in multiple sclerosis lesions as detected by flow cytometry on rapid autopsy derived brain tissue from multiple sclerosis patients. Collectively, our findings reveal that MCAM is at the centre of a pathological pathway used by brain endothelial cells to recruit pathogenic CD4+ T lymphocyte from circulation early during neuroinflammation. The therapeutic targeting of this mechanism is a promising avenue to treat multiple sclerosis.
Asunto(s)
Encefalomielitis Autoinmune Experimental , Esclerosis Múltiple , Humanos , Barrera Hematoencefálica/patología , Encéfalo/patología , Antígeno CD146/metabolismo , Linfocitos T CD4-Positivos/metabolismo , Encefalomielitis Autoinmune Experimental/patología , Células Endoteliales/metabolismo , Endotelio/metabolismo , Endotelio/patología , Esclerosis Múltiple/patología , Enfermedades NeuroinflamatoriasRESUMEN
B cells have been implicated in the pathogenesis of multiple sclerosis, but the mechanisms that guide B cell activation in the periphery and subsequent migration to the CNS remain incompletely understood. We previously showed that systemic inflammation induces an accumulation of B cells in the spleen in a CCR6/CCL20-dependent manner. In this study, we evaluated the role of CCR6/CCL20 in the context of myelin oligodendrocyte glycoprotein (MOG) protein-induced (B cell-dependent) experimental autoimmune encephalomyelitis (EAE). We found that CCR6 is upregulated on murine B cells that migrate into the CNS during neuroinflammation. In addition, human B cells that migrate across CNS endothelium in vitro were found to be CCR6+, and we detected CCL20 production by activated CNS-derived human endothelial cells as well as a systemic increase in CCL20 protein during EAE. Although mice that lack CCR6 expression specifically on B cells exhibited an altered germinal center reaction in response to MOG protein immunization, CCR6-deficient B cells did not exhibit any competitive disadvantage in their migration to the CNS during EAE, and the clinical and pathological presentation of EAE induced by MOG protein was unaffected. These data, to our knowledge, provide new information on the role of B cell-intrinsic CCR6 expression in a B cell-dependent model of neuroinflammation.
Asunto(s)
Linfocitos B/inmunología , Encefalomielitis Autoinmune Experimental/inmunología , Centro Germinal/inmunología , Inmunización/métodos , Glicoproteína Mielina-Oligodendrócito/administración & dosificación , Receptores CCR6/deficiencia , Animales , Linfocitos B/metabolismo , Donantes de Sangre , Barrera Hematoencefálica/citología , Barrera Hematoencefálica/inmunología , Movimiento Celular/genética , Movimiento Celular/inmunología , Células Cultivadas , Quimiocina CCL20/metabolismo , Encefalomielitis Autoinmune Experimental/inducido químicamente , Células Endoteliales/inmunología , Femenino , Humanos , Ratones , Ratones Endogámicos C57BL , Ratones Noqueados , Glicoproteína Mielina-Oligodendrócito/genética , Receptores CCR6/genética , Proteínas Recombinantes/administración & dosificaciónRESUMEN
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.
Asunto(s)
Molécula de Adhesión Celular del Leucocito Activado/metabolismo , Encefalomielitis Autoinmune Experimental/metabolismo , Células Endoteliales/metabolismo , Molécula de Adhesión Celular del Leucocito Activado/genética , Animales , Barrera Hematoencefálica/citología , Barrera Hematoencefálica/metabolismo , Células Cultivadas , Encefalomielitis Autoinmune Experimental/patología , Femenino , Homeostasis , Ratones Endogámicos C57BL , Ratones Noqueados , Glicoproteína Mielina-Oligodendrócito , Fragmentos de Péptidos , Índice de Severidad de la Enfermedad , Médula Espinal/metabolismo , Proteínas de Uniones Estrechas/metabolismoRESUMEN
Blood-brain barrier function is driven by the influence of astrocyte-secreted factors. During neuroinflammatory responses the blood-brain barrier is compromised resulting in central nervous system damage and exacerbated pathology. Here, we identified endothelial netrin 1 induction as a vascular response to astrocyte-derived sonic hedgehog that promotes autocrine barrier properties during homeostasis and increases with inflammation. Netrin 1 supports blood-brain barrier integrity by upregulating endothelial junctional protein expression, while netrin 1 knockout mice display disorganized tight junction protein expression and barrier breakdown. Upon inflammatory conditions, blood-brain barrier endothelial cells significantly upregulated netrin 1 levels in vitro and in situ, which prevented junctional breach and endothelial cell activation. Finally, netrin 1 treatment during experimental autoimmune encephalomyelitis significantly reduced blood-brain barrier disruption and decreased clinical and pathological indices of disease severity. Our results demonstrate that netrin 1 is an important regulator of blood-brain barrier maintenance that protects the central nervous system against inflammatory conditions such as multiple sclerosis and experimental autoimmune encephalomyelitis.
Asunto(s)
Barrera Hematoencefálica/metabolismo , Encefalomielitis Autoinmune Experimental/tratamiento farmacológico , Encefalomielitis Autoinmune Experimental/metabolismo , Inflamación/metabolismo , Esclerosis Múltiple/metabolismo , Factores de Crecimiento Nervioso/fisiología , Factores de Crecimiento Nervioso/uso terapéutico , Proteínas Supresoras de Tumor/fisiología , Proteínas Supresoras de Tumor/uso terapéutico , Animales , Antiinflamatorios/farmacología , Antiinflamatorios/uso terapéutico , Proteínas Sanguíneas/metabolismo , Barrera Hematoencefálica/efectos de los fármacos , Células Endoteliales/metabolismo , Humanos , Inflamación/tratamiento farmacológico , Mediadores de Inflamación/metabolismo , Proteínas de la Membrana/metabolismo , Ratones , Ratones Noqueados , Factores de Crecimiento Nervioso/genética , Factores de Crecimiento Nervioso/metabolismo , Factores de Crecimiento Nervioso/farmacología , Netrina-1 , Permeabilidad , Cultivo Primario de Células , Uniones Estrechas/metabolismo , Proteínas Supresoras de Tumor/genética , Proteínas Supresoras de Tumor/metabolismo , Proteínas Supresoras de Tumor/farmacología , Regulación hacia ArribaRESUMEN
Early changes in the normal appearing white matter of multiple sclerosis (MS) patients precede the appearance of gadolinium-enhancing lesions. Although these findings suggest blood-brain barrier (BBB) breakdown as an important feature in MS pathogenesis, limited information is available on the BBB alterations during lesion genesis. Here, we perform a longitudinal characterization of the vascular, neuropathological and immunological changes before lesion formation in mice developing spontaneous relapsing-remitting experimental autoimmune encephalomyelitis (sRR-EAE). We found a significant upregulation of Th1 and Th17 cytokines in the periphery of sRR-EAE mice before any evident neuropathology. In the CNS, BBB and astroglial activations were the first pathological changes occurring after 45days of age and were followed by immune cell infiltration by day 50. These pathological alterations subsequently led to perivascular demyelination and disease onset. In MS, (p)reactive lesions mirrored the changes seen in early sRR-EAE by displaying considerable BBB disruption, perivascular astrogliosis, redistribution of junctional proteins and increased expression of endothelial cell adhesion molecules. Our findings suggest that BBB breach occurs before significant immune cell infiltration and demyelination. In addition, peripheral immune activation during sRR-EAE precedes CNS pathology, suggesting that outside in signaling mechanisms play a role in the development of neuroinflammatory lesions.
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Barrera Hematoencefálica/metabolismo , Encefalomielitis Autoinmune Experimental/metabolismo , Esclerosis Múltiple Crónica Progresiva/metabolismo , Esclerosis Múltiple Recurrente-Remitente/metabolismo , Adulto , Anciano , Animales , Astrocitos/metabolismo , Astrocitos/patología , Barrera Hematoencefálica/patología , Encéfalo/metabolismo , Encéfalo/patología , Encefalomielitis Autoinmune Experimental/patología , Endotelio Vascular/metabolismo , Endotelio Vascular/patología , Femenino , Gliosis/metabolismo , Gliosis/patología , Humanos , Estudios Longitudinales , Masculino , Ratones Transgénicos , Persona de Mediana Edad , Esclerosis Múltiple Crónica Progresiva/patología , Esclerosis Múltiple Recurrente-Remitente/patología , Linfocitos T/metabolismoRESUMEN
In multiple sclerosis, encephalitogenic CD4(+) lymphocytes require adhesion molecules to accumulate into central nervous system inflammatory lesions. Using proteomic techniques, we identified expression of melanoma cell adhesion molecule (MCAM) on a subset of human effector memory CD4(+) lymphocytes and on human blood-brain barrier endothelium. Herein, we demonstrate that MCAM is a stable surface marker that refines the identification of interleukin 17(+), interleukin 22(+), RAR-related orphan receptor γ and interleukin 23 receptor(+) cells within the CD161(+)CCR6(+) subset of memory CD4(+) lymphocytes. We also show that MCAM(+) lymphocytes express significantly more granulocyte/macrophage colony stimulating factor and granzyme B than MCAM(-) lymphocytes. Furthermore, the proportion of MCAM(+) CD4(+) lymphocytes is significantly increased in the blood and in the central nervous system of patients with multiple sclerosis and experimental autoimmune encephalomyelitis animals compared with healthy controls or other neurological diseases, and MCAM expression is upregulated at the blood-brain barrier within inflammatory lesions. Moreover, blockade of MCAM or depletion of MCAM(+) CD4(+) T lymphocytes both restrict the migration of T(H)17 lymphocytes across blood-brain barrier endothelial cells and decrease the severity of experimental autoimmune encephalomyelitis. Our findings indicate that MCAM could serve as a potential biomarker for multiple sclerosis and represents a valuable target for the treatment of neuroinflammatory conditions.
Asunto(s)
Movimiento Celular/inmunología , Sistema Nervioso Central/inmunología , Sistema Nervioso Central/metabolismo , Encefalomielitis Autoinmune Experimental/inmunología , Encefalomielitis Autoinmune Experimental/metabolismo , Animales , Biomarcadores/metabolismo , Antígeno CD146/metabolismo , Antígeno CD146/fisiología , Células Cultivadas , Sistema Nervioso Central/patología , Encefalomielitis Autoinmune Experimental/patología , Humanos , Ratones , Ratones Endogámicos C57BL , Esclerosis Múltiple/inmunología , Esclerosis Múltiple/metabolismo , Esclerosis Múltiple/patología , Inflamación Neurogénica/inmunología , Inflamación Neurogénica/metabolismo , Inflamación Neurogénica/patologíaRESUMEN
BACKGROUND AND OBJECTIVES: Multiple sclerosis (MS) is a neuroinflammatory and neurodegenerative disease characterized by infiltration of immune cells in multifocal areas of the CNS. The specific molecular processes allowing autoreactive immune cells to enter the CNS compartment through the blood-brain barrier remain elusive. METHODS: Using endothelial cell (EC) enrichment and single-cell RNA sequencing, we characterized the cells implicated in the neuroinflammatory processes in experimental autoimmune encephalomyelitis, an animal model of MS. Validations on human MS brain sections of the most differentially expressed genes in venous ECs were performed using immunohistochemistry and confocal microscopy. RESULTS: We found an upregulation of genes associated with antigen presentation and interferon in most populations of CNS-resident cells, including ECs. Interestingly, instead of transcriptionally distinct profiles, a continuous gradient of gene expression separated the arteriovenous zonation of the brain vasculature. However, differential gene expression analysis presented more transcriptomic alterations on the venous side of the axis, suggesting a prominent role of venous ECs in neuroinflammation. Furthermore, analysis of ligand-receptor interactions identified important potential molecular communications between venous ECs and infiltrated immune populations. To confirm the relevance of our observation in the context of human disease, we validated the protein expression of the most upregulated genes (Ackr1 and Lcn2) in MS lesions. DISCUSSION: In this study, we provide a landscape of the cellular heterogeneity associated with neuroinflammation. We also present important molecular insights for further exploration of specific cell processes that promote infiltration of immune cells inside the brain of experimental autoimmune encephalomyelitis mice.
Asunto(s)
Encefalitis , Encefalomielitis Autoinmune Experimental , Esclerosis Múltiple , Enfermedades Neurodegenerativas , Humanos , Animales , Ratones , Encefalomielitis Autoinmune Experimental/genética , Transcriptoma , Esclerosis Múltiple/genética , Encéfalo , EndotelioRESUMEN
OBJECTIVE: Blood-derived myeloid antigen-presenting cells (APCs) account for a significant proportion of the leukocytes found within lesions of multiple sclerosis (MS) and experimental allergic encephalomyelitis (EAE). These APCs along with activated microglia are thought to be pivotal in the initiation of the central nervous system (CNS)-targeted immune response in MS and EAE. However, the exact molecules that direct the migration of myeloid cells from the periphery across the blood-brain barrier (BBB) remain largely unknown. METHODS: We identified Ninjurin-1 in a proteomic screen of human BBB endothelial cells (ECs). We assessed the expression of Ninjurin-1 by BBB-ECs and immune cells, and we determined the role of Ninjurin-1 in immune cell migration to the CNS in vivo in EAE mice. RESULTS: Ninjurin-1 was found to be weakly expressed in the healthy human and mouse CNS but upregulated on BBB-ECs and on infiltrating APCs during the course of EAE and in active MS lesions. In human peripheral blood, Ninjurin-1 was predominantly expressed by monocytes, whereas it was barely detectable on T and B lymphocytes. Moreover, Ninjurin-1 neutralization specifically abrogated the adhesion and migration of human monocytes across BBB-ECs, without affecting lymphocyte recruitment. Finally, Ninjurin-1 blockade reduced clinical disease activity and histopathological indices of EAE and decreased infiltration of macrophages, dendritic cells, and APCs into the CNS. INTERPRETATION: Our study uncovers an important cell-specific role for Ninjurin-1 in the transmigration of inflammatory APCs across the BBB and further emphasizes the importance of myeloid cell recruitment during the development of neuroinflammatory lesions.
Asunto(s)
Moléculas de Adhesión Celular Neuronal/metabolismo , Movimiento Celular/fisiología , Sistema Nervioso Central/inmunología , Encefalomielitis Autoinmune Experimental/metabolismo , Células Mieloides/citología , Células Mieloides/metabolismo , Factores de Crecimiento Nervioso/metabolismo , Animales , Linfocitos B/metabolismo , Modelos Animales de Enfermedad , Femenino , Humanos , Ratones , Ratones Endogámicos C57BL , Monocitos/metabolismo , Linfocitos T/metabolismoRESUMEN
Clonally expanded CD8(+) T lymphocytes are present in multiple sclerosis lesions, as well as in the cerebrospinal fluid of patients with multiple sclerosis. In experimental autoimmune encephalomyelitis, CD8(+) T lymphocytes are found in spinal cord and brainstem lesions. However, the exact phenotype of central nervous system-infiltrating CD8(+) T lymphocytes and the mechanism by which these cells cross the blood-brain barrier remain largely unknown. Using cerebrospinal fluid from patients with multiple sclerosis, spinal cord from experimental autoimmune encephalomyelitis and coronavirus-induced encephalitis, we demonstrate that central nervous system-infiltrating CD8(+) T lymphocytes are mostly of the effector memory phenotype (CD62L(-) CCR7(-) granzymeB(hi)). We further show that purified human effector memory CD8(+) T lymphocytes transmigrate more readily across blood-brain barrier-endothelial cells than non-effector memory CD8(+) T lymphocytes, and that blood-brain barrier endothelium promotes the selective recruitment of effector memory CD8(+) T lymphocytes. Furthermore, we provide evidence for the recruitment of interferon-γ- and interleukin-17-secreting CD8(+) T lymphocytes by human and mouse blood-brain barrier endothelium. Finally, we show that in vitro migration of CD8(+) T lymphocytes across blood-brain barrier-endothelial cells is dependent on α4 integrin, but independent of intercellular adhesion molecule-1/leucocyte function-associated antigen-1, activated leucocyte cell adhesion molecule/CD6 and the chemokine monocyte chemotactic protein-1/CCL2. We also demonstrate that in vivo neutralization of very late antigen-4 restricts central nervous system infiltration of CD8(+) T lymphocytes in active immunization and adoptive transfer experimental autoimmune encephalomyelitis, and in coronavirus-induced encephalitis. Our study thus demonstrates an active role of the blood-brain barrier in the recruitment of effector memory CD8(+) T lymphocytes to the CNS compartment and defines α4 integrin as a major contributor of CD8(+) T lymphocyte entry into the brain.
Asunto(s)
Linfocitos T CD8-positivos/inmunología , Infecciones por Coronavirus/inmunología , Encefalitis Viral/inmunología , Encefalomielitis Autoinmune Experimental/inmunología , Integrina alfa4/metabolismo , Esclerosis Múltiple/inmunología , Adulto , Animales , Barrera Hematoencefálica/inmunología , Barrera Hematoencefálica/metabolismo , Linfocitos T CD8-positivos/metabolismo , Movimiento Celular/inmunología , Infecciones por Coronavirus/metabolismo , Encefalitis Viral/metabolismo , Encefalomielitis Autoinmune Experimental/metabolismo , Humanos , Memoria Inmunológica , Ratones , Persona de Mediana Edad , Esclerosis Múltiple/metabolismoRESUMEN
BACKGROUND AND OBJECTIVES: In multiple sclerosis (MS), peripheral immune cells use various cell trafficking molecules to infiltrate the CNS where they cause damage.The objective of this study was to investigate the involvement of coxsackie and adenovirus receptor-like membrane protein (CLMP) in the migration of immune cells into the CNS of patients with MS. METHODS: Expression of CLMP was measured in primary cultures of human brain endothelial cells (HBECs) and human meningeal endothelial cells (HMECs), postmortem brain samples, and peripheral blood mononuclear cells (PBMCs) from patients with MS and controls by RNA sequencing, quantitative PCR, immunohistochemistry, and flow cytometry. In vitro migration assays using HBECs and HMECs were performed to evaluate the function of CLMP. RESULTS: Using bulk RNA sequencing of primary cultures of human brain and meningeal endothelial cells (ECs), we have identified CLMP as a new potential cell trafficking molecule upregulated in inflammatory conditions. We first confirmed the upregulation of CLMP at the protein level on TNFα-activated and IFNγ-activated primary cultures of human brain and meningeal ECs. In autopsy brain specimens from patients with MS, we demonstrated an overexpression of endothelial CLMP in active MS lesions when compared with normal control brain tissue. Flow cytometry of human PBMCs demonstrated an increased frequency of CLMP+ B lymphocytes and monocytes in patients with MS, when compared with that in healthy controls. The use of a blocking antibody against CLMP reduced the migration of immune cells across the human brain and meningeal ECs in vitro. Finally, we found CLMP+ immune cell infiltrates in the perivascular area of parenchymal lesions and in the meninges of patients with MS. DISCUSSION: Collectively, our data demonstrate that CLMP is an adhesion molecule used by immune cells to access the CNS during neuroinflammatory disorders such as MS. CLMP could represent a target for a new treatment of neuroinflammatory conditions.
Asunto(s)
Esclerosis Múltiple , Humanos , Encéfalo/metabolismo , Proteína de la Membrana Similar al Receptor de Coxsackie y Adenovirus/metabolismo , Células Endoteliales/metabolismo , Leucocitos/metabolismo , Leucocitos Mononucleares , Factor de Necrosis Tumoral alfa/metabolismoRESUMEN
The migration of circulating leukocytes into the central nervous system (CNS) is a key driver of multiple sclerosis (MS) pathogenesis. The monoclonal antibody natalizumab proved that pharmaceutically obstructing this process is an effective therapeutic approach for treating relapsing-remitting MS (RRMS). Unfortunately, the clinical efficacy of natalizumab is somewhat offset by its incapacity to control the progressive forms of MS (PMS) and by life-threatening side effects in RRMS rising from the expression of its molecular target, very late antigen 4 (VLA4), on most immune cells and consequent impairment of CNS immunosurveillance. Here, we identified dual immunoglobulin domain containing cell adhesion molecule (DICAM) as a cell trafficking molecule preferentially expressed by T helper 17 (TH17)polarized CD4+ T lymphocytes. We found that DICAM expression on circulating CD4+ T cells was increased in patients with active RRMS and PMS disease courses, and expression of DICAM ligands was increased on the blood-brain barrier endothelium upon inflammation and in MS lesions. Last, we demonstrated that pharmaceutically neutralizing DICAM reduced murine and human TH17 cell trafficking across the blood-brain barrier in vitro and in vivo, and alleviated disease symptoms in four distinct murine autoimmune encephalomyelitis models, including relapsing-remitting and progressive disease models. Collectively, our data highlight DICAM as a candidate therapeutic target to impede the migration of disease-inducing leukocytes into the CNS in both RRMS and PMS and suggest that blocking DICAM with a monoclonal antibody may be a promising therapeutic approach.
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Esclerosis Múltiple Recurrente-Remitente , Esclerosis Múltiple , Animales , Barrera Hematoencefálica/metabolismo , Moléculas de Adhesión Celular/metabolismo , Humanos , Ratones , Esclerosis Múltiple/tratamiento farmacológico , Esclerosis Múltiple/metabolismo , Natalizumab/metabolismo , Natalizumab/farmacología , Natalizumab/uso terapéutico , Enfermedades Neuroinflamatorias , Linfocitos T/metabolismo , Células Th17RESUMEN
Clathrin-coated vesicles (CCVs) are major carriers for endocytic cargo and mediate important intracellular trafficking events at the trans-Golgi network (TGN) and endosomes. Whereas clathrin heavy chain provides the structural backbone of the clathrin coat, the role of clathrin light chains (CLCs) is poorly understood. We now demonstrate that CLCs are not required for clathrin-mediated endocytosis but are critical for clathrin-mediated trafficking between the TGN and the endosomal system. Specifically, CLC knockdown (KD) causes the cation-independent mannose-6 phosphate receptor (CI-MPR) to cluster near the TGN leading to a delay in processing of the lysosomal hydrolase cathepsin D. A recently identified binding partner for CLCs is huntingtin-interacting protein 1-related (HIP1R), which is required for productive interactions of CCVs with the actin cytoskeleton. CLC KD causes mislocalization of HIP1R and overassembly of actin, which accumulates in patches around the clustered CI-MPR. A dominant-negative CLC construct that disrupts HIP1R/CLC interactions causes similar alterations in CI-MPR trafficking and actin assembly. Thus, in mammalian cells CLCs function in intracellular membrane trafficking by acting as recruitment proteins for HIP1R, enabling HIP1R to regulate actin assembly on clathrin-coated structures.
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Actinas/química , Cadenas Ligeras de Clatrina/fisiología , Lectinas Tipo C/metabolismo , Lectinas de Unión a Manosa/metabolismo , Receptores de Superficie Celular/metabolismo , Animales , Transporte Biológico , Antígenos CD8/biosíntesis , Células COS , Catepsina D/química , Catepsina D/metabolismo , Chlorocebus aethiops , Cadenas Ligeras de Clatrina/química , Endocitosis , Aparato de Golgi/metabolismo , Células HeLa , Humanos , Receptor de Manosa , Modelos Biológicos , Proteínas del Tejido Nervioso/química , ARN Interferente Pequeño/metabolismo , Red trans-GolgiRESUMEN
Dysregulated immune profiles have been described in symptomatic patients infected with SARS-CoV-2. Whether the reported immune alterations are specific to SARS-CoV-2 infection or also triggered by other acute illnesses remains unclear. We performed flow cytometry analysis on fresh peripheral blood from a consecutive cohort of (a) patients hospitalized with acute SARS-CoV-2 infection, (b) patients of comparable age and sex hospitalized for another acute disease (SARS-CoV-2 negative), and (c) healthy controls. Using both data-driven and hypothesis-driven analyses, we found several dysregulations in immune cell subsets (e.g., decreased proportion of T cells) that were similarly associated with acute SARS-CoV-2 infection and non-COVID-19-related acute illnesses. In contrast, we identified specific differences in myeloid and lymphocyte subsets that were associated with SARS-CoV-2 status (e.g., elevated proportion of ICAM-1+ mature/activated neutrophils, ALCAM+ monocytes, and CD38+CD8+ T cells). A subset of SARS-CoV-2-specific immune alterations correlated with disease severity, disease outcome at 30 days, and mortality. Our data provide an understanding of the immune dysregulation specifically associated with SARS-CoV-2 infection among acute care hospitalized patients. Our study lays the foundation for the development of specific biomarkers to stratify SARS-CoV-2-positive patients at risk of unfavorable outcomes and to uncover candidate molecules to investigate from a therapeutic perspective.
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COVID-19/inmunología , Leucocitos/clasificación , Leucocitos/inmunología , SARS-CoV-2 , Enfermedad Aguda , Adulto , Anciano , Subgrupos de Linfocitos B/inmunología , Linfocitos T CD4-Positivos/inmunología , Linfocitos T CD8-positivos/inmunología , COVID-19/epidemiología , COVID-19/mortalidad , Estudios de Casos y Controles , Estudios de Cohortes , Femenino , Hospitalización , Humanos , Activación de Linfocitos , Masculino , Persona de Mediana Edad , Modelos Inmunológicos , Monocitos/inmunología , Análisis Multivariante , Neutrófilos/inmunología , Pandemias , Pronóstico , Estudios Prospectivos , Quebec/epidemiología , Factores de Riesgo , SARS-CoV-2/inmunología , Índice de Severidad de la EnfermedadRESUMEN
The authors wish to make the following corrections to this paper [...].
RESUMEN
OBJECTIVE: To investigate the involvement of interleukin (IL)-26 in neuroinflammatory processes in multiple sclerosis (MS), in particular in blood-brain barrier (BBB) integrity. METHODS: Expression of IL-26 was measured in serum, CSF, in vitro differentiated T helper (TH) cell subsets, and postmortem brain tissue of patients with MS and controls by ELISA, quantitative PCR, and immunohistochemistry. Primary human and mouse BBB endothelial cells (ECs) were treated with IL-26 in vitro and assessed for BBB integrity. RNA sequencing was performed on IL-26-treated human BBB ECs. Myelin oligodendrocyte glycoprotein35-55 experimental autoimmune encephalomyelitis (EAE) mice were injected IP with IL-26. BBB leakage and immune cell infiltration were assessed in the CNS of these mice using immunohistochemistry and flow cytometry. RESULTS: IL-26 expression was induced in TH lymphocytes by TH17-inducing cytokines and was upregulated in the blood and CSF of patients with MS. CD4+IL-26+ T lymphocytes were found in perivascular infiltrates in MS brain lesions, and both receptor chains for IL-26 (IL-10R2 and IL-20R1) were detected on BBB ECs in vitro and in situ. In contrast to IL-17 and IL-22, IL-26 promoted integrity and reduced permeability of BBB ECs in vitro and in vivo. In EAE, IL-26 reduced disease severity and proinflammatory lymphocyte infiltration into the CNS, while increasing infiltration of Tregs. CONCLUSIONS: Our study demonstrates that although IL-26 is preferentially expressed by TH17 lymphocytes, it promotes BBB integrity in vitro and in vivo and is protective in chronic EAE, highlighting the functional diversity of cytokines produced by TH17 lymphocytes.
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Barrera Hematoencefálica/metabolismo , Encefalomielitis Autoinmune Experimental/metabolismo , Interleucinas/metabolismo , Esclerosis Múltiple/metabolismo , Células Th17/metabolismo , Animales , Células Cultivadas , Encefalomielitis Autoinmune Experimental/tratamiento farmacológico , Endotelio Vascular/metabolismo , Feto , Humanos , Interleucinas/sangre , Interleucinas/líquido cefalorraquídeo , Interleucinas/farmacología , Ratones , Esclerosis Múltiple/sangre , Esclerosis Múltiple/líquido cefalorraquídeoRESUMEN
Since most preclinical drug permeability assays across the blood-brain barrier (BBB) are still evaluated in rodents, we compared an in vitro mouse primary endothelial cell model to the mouse b.End3 and the acellular parallel artificial membrane permeability assay (PAMPA) models for drug screening purposes. The mRNA expression of key feature membrane proteins of primary and bEnd.3 mouse brain endothelial cells were compared. Transwell® monolayer models were further characterized in terms of tightness and integrity. The in vitro in vivo correlation (IVIVC) was obtained by the correlation of the in vitro permeability data with log BB values obtained in mice for seven drugs. The mouse primary model showed higher monolayer integrity and levels of mRNA expression of BBB tight junction (TJ) proteins and membrane transporters (MBRT), especially for the efflux transporter Pgp. The IVIVC and drug ranking underlined the superiority of the primary model (r2 = 0.765) when compared to the PAMPA-BBB (r2 = 0.391) and bEnd.3 cell line (r2 = 0.019) models. The primary monolayer mouse model came out as a simple and reliable candidate for the prediction of drug permeability across the BBB. This model encompasses a rapid set-up, a fair reproduction of BBB tissue characteristics, and an accurate drug screening.
RESUMEN
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.
Asunto(s)
Astrocitos , Encéfalo/citología , Separación Celular , Células Endoteliales , Pericitos , Animales , Barrera Hematoencefálica/citología , Técnicas de Cultivo de Célula , Femenino , Citometría de Flujo , Masculino , Ratones , Ratones Endogámicos C57BL , Microscopía ConfocalRESUMEN
TCR1640 mice, which have a T cell receptor (TCR) directed against MOG92-106, spontaneously develop experimental autoimmune encephalomyelitis. Female mice mostly develop a relapsing-remitting (RR) course and have a higher incidence of disease, while males most frequently suffer from progressive disease, reflecting the unresolved clinical sex discrepancies seen in multiple sclerosis. Herein, we performed adoptive transfers of male and female TCR1640 immune cells into WT animals to investigate if disease course is dependent on the sex of the donor immune cells or on the sex of the recipient animal. We found that transfer of female TCR1640 immune cells led to a RR disease while transfer of male TCR1640 immune cells led to a progressive course, independent of the sex of the recipient. In addition, regulatory and pathogenic T cell infiltration after transfer was also immune cell sex intrinsic. We performed genetic profiling of the donor immune cells and found significant differences between the transcriptomic profiles of male and female TCR1640 immune cells, interestingly, within genes related to immune regulation of T lymphocytes. These results suggest that differences in gene expression profiles related to regulation of T cell immunity seen in male and female neuroinflammatory disease drive relapsing versus progressive disease course.
Asunto(s)
Enfermedades Desmielinizantes/genética , Enfermedades Desmielinizantes/inmunología , Progresión de la Enfermedad , Receptores de Antígenos de Linfocitos T/inmunología , Traslado Adoptivo , Animales , Enfermedades Autoinmunes/inmunología , Barrera Hematoencefálica/patología , Modelos Animales de Enfermedad , Femenino , Factores Inmunológicos , Masculino , Ratones , Ratones Transgénicos , Esclerosis Múltiple/inmunología , Fenotipo , Receptores de Antígenos de Linfocitos T/metabolismo , Recurrencia , Factores Sexuales , Linfocitos T/inmunología , TranscriptomaRESUMEN
The presence of B lymphocyte-associated oligoclonal immunoglobulins in the cerebrospinal fluid is a classic hallmark of multiple sclerosis (MS). The clinical efficacy of anti-CD20 therapies supports a major role for B lymphocytes in MS development. Although activated oligoclonal populations of pathogenic B lymphocytes are able to traffic between the peripheral circulation and the central nervous system (CNS) in patients with MS, molecular players involved in this migration have not yet been elucidated. In this study, we demonstrated that activated leukocyte cell adhesion molecule (ALCAM/CD166) identifies subsets of proinflammatory B lymphocytes and drives their transmigration across different CNS barriers in mouse and human. We also showcased that blocking ALCAM alleviated disease severity in animals affected by a B cell-dependent form of experimental autoimmune encephalomyelitis. Last, we determined that the proportion of ALCAM+ B lymphocytes was increased in the peripheral blood and within brain lesions of patients with MS. Our findings indicate that restricting access to the CNS by targeting ALCAM on pathogenic B lymphocytes might represent a promising strategy for the development of next-generation B lymphocyte-targeting therapies for the treatment of MS.
Asunto(s)
Molécula de Adhesión Celular del Leucocito Activado/metabolismo , Linfocitos B/citología , Movimiento Celular , Sistema Nervioso Central/metabolismo , Animales , Barrera Hematoencefálica/metabolismo , Encefalomielitis Autoinmune Experimental/inmunología , Encefalomielitis Autoinmune Experimental/patología , Endotelio/metabolismo , Humanos , Memoria Inmunológica , Ratones Noqueados , Esclerosis Múltiple/inmunología , Esclerosis Múltiple/patología , Glicoproteína Mielina-Oligodendrócito/inmunología , Proteínas Recombinantes/inmunología , Índice de Severidad de la EnfermedadRESUMEN
CD70 is the unique ligand of CD27 and is expressed on immune cells only upon activation. Therefore, engagement of the costimulatory CD27/CD70 pathway is solely dependent on upregulation of CD70. However, the T cell-intrinsic effect and function of human CD70 remain underexplored. Herein, we describe that CD70 expression distinguishes proinflammatory CD4+ T lymphocytes that display an increased potential to migrate into the central nervous system (CNS). Upregulation of CD70 on CD4+ T lymphocytes is induced by TGF-ß1 and TGF-ß3, which promote a pathogenic phenotype. In addition, CD70 is associated with a TH1 and TH17 profile of lymphocytes and is important for T-bet and IFN-γ expression by both T helper subtypes. Moreover, adoptive transfer of CD70-/-CD4+ T lymphocytes induced less severe experimental autoimmune encephalomyelitis (EAE) disease than transfer of WT CD4+ T lymphocytes. CD70+CD4+ T lymphocytes are found in the CNS during acute autoimmune inflammation in humans and mice, highlighting CD70 as both an immune marker and an important costimulator of highly pathogenic proinflammatory TH1/TH17 lymphocytes infiltrating the CNS.