RESUMEN
BACKGROUND: Severe rebound multiple sclerosis (MS) activity is a life-threatening complication of natalizumab (NTZ) withdrawal, for which pathogenesis and treatment are still unclear. We report the immunological and pathological characterization of a case of central nervous system (CNS) inflammatory demyelination after NTZ discontinuation. OBJECTIVE: To understand the pathophysiology of this neuroinflammatory condition. METHODS: Antemortem blood and cerebrospinal fluid (CSF) analysis was compared with postmortem pathological studies, as well as with novel flow cytometry characterization of immune cells isolated from the CNS parenchyma. RESULTS: Pathological analysis of the brain revealed the presence of innumerable active inflammatory demyelinating lesions typical of immunopathological pattern II. Monocytes/macrophages and B cells were enriched in the CNS parenchyma compared to the CSF. Numerous plasma cells were present in the lesions, but CD8 T lymphocytes were predominant in the parenchyma, as opposed to CD4 in the CSF. CNS-infiltrating lymphocytes expressed high levels of adhesion molecules, granzyme B (GzB), interferon-gamma (IFN-γ), and interleukin (IL)-17. CONCLUSIONS: Our results underline the differences in immune cell populations between the CSF and the CNS parenchyma, and suggest that aggressive immunosuppressive therapy targeting both T and B lymphocytes is warranted to control the overwhelming CNS inflammation.
Asunto(s)
Linfocitos B/patología , Esclerosis Múltiple/tratamiento farmacológico , Esclerosis Múltiple/patología , Natalizumab/uso terapéutico , Síndrome de Abstinencia a Sustancias/patología , Linfocitos T/patología , Adulto , Linfocitos B/inmunología , Femenino , Humanos , Interferón gamma/uso terapéutico , Esclerosis Múltiple/diagnóstico por imagen , Linfocitos T/inmunologíaRESUMEN
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.
Asunto(s)
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
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.
Asunto(s)
Endotelio Vascular/metabolismo , Proteínas Fluorescentes Verdes/genética , Microvasos/metabolismo , Mitocondrias/metabolismo , Animales , Secuencia de Bases , Cartilla de ADN , Endotelio Vascular/citología , Citometría de Flujo , Colorantes Fluorescentes , Ratones , Ratones Transgénicos , Microvasos/citología , Reacción en Cadena de la PolimerasaRESUMEN
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
The blood-brain barrier (BBB) is composed of tightly bound endothelial cells (ECs) and perivascular astrocytes that regulate central nervous system (CNS) homeostasis. We showed that astrocytes secrete Sonic hedgehog and that BBB ECs express Hedgehog (Hh) receptors, which together promote BBB formation and integrity during embryonic development and adulthood. Using pharmacological inhibition and genetic inactivation of the Hh signaling pathway in ECs, we also demonstrated a critical role of the Hh pathway in promoting the immune quiescence of BBB ECs by decreasing the expression of proinflammatory mediators and the adhesion and migration of leukocytes, in vivo and in vitro. Overall, the Hh pathway provides a barrier-promoting effect and an endogenous anti-inflammatory balance to CNS-directed immune attacks, as occurs in multiple sclerosis.
Asunto(s)
Astrocitos/metabolismo , Barrera Hematoencefálica/fisiología , Encéfalo/inmunología , Células Endoteliales/metabolismo , Proteínas Hedgehog/metabolismo , Transducción de Señal , Animales , Barrera Hematoencefálica/citología , Encéfalo/fisiología , Linfocitos T CD4-Positivos/fisiología , Adhesión Celular , Movimiento Celular , Células Cultivadas , Quimiocinas/metabolismo , Impedancia Eléctrica , Encefalomielitis Autoinmune Experimental/inmunología , Encefalomielitis Autoinmune Experimental/metabolismo , Femenino , Proteínas Hedgehog/genética , Humanos , Mediadores de Inflamación/metabolismo , Ratones , Ratones Endogámicos C57BL , Esclerosis Múltiple/inmunología , Esclerosis Múltiple/metabolismo , Permeabilidad , Alcaloides de Veratrum/farmacologíaRESUMEN
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.