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1.
Brain ; 146(4): 1483-1495, 2023 04 19.
Artigo em Inglês | MEDLINE | ID: mdl-36319587

RESUMO

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.


Assuntos
Encefalomielite Autoimune Experimental , Esclerose Múltipla , Humanos , Barreira Hematoencefálica/patologia , Encéfalo/patologia , Antígeno CD146/metabolismo , Linfócitos T CD4-Positivos/metabolismo , Encefalomielite Autoimune Experimental/patologia , Células Endoteliais/metabolismo , Endotélio/metabolismo , Endotélio/patologia , Esclerose Múltipla/patologia , Doenças Neuroinflamatórias
2.
Sci Transl Med ; 14(626): eabj0473, 2022 01 05.
Artigo em Inglês | MEDLINE | ID: mdl-34985970

RESUMO

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.


Assuntos
Esclerose Múltipla Recidivante-Remitente , Esclerose Múltipla , Animais , Barreira Hematoencefálica/metabolismo , Moléculas de Adesão Celular/metabolismo , Humanos , Camundongos , Esclerose Múltipla/tratamento farmacológico , Esclerose Múltipla/metabolismo , Natalizumab/metabolismo , Natalizumab/farmacologia , Natalizumab/uso terapêutico , Doenças Neuroinflamatórias , Linfócitos T/metabolismo , Células Th17
3.
Artigo em Inglês | MEDLINE | ID: mdl-32788322

RESUMO

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.


Assuntos
Barreira Hematoencefálica/metabolismo , Encefalomielite Autoimune Experimental/metabolismo , Interleucinas/metabolismo , Esclerose Múltipla/metabolismo , Células Th17/metabolismo , Animais , Células Cultivadas , Encefalomielite Autoimune Experimental/tratamento farmacológico , Endotélio Vascular/metabolismo , Feto , Humanos , Interleucinas/sangue , Interleucinas/líquido cefalorraquidiano , Interleucinas/farmacologia , Camundongos , Esclerose Múltipla/sangue , Esclerose Múltipla/líquido cefalorraquidiano
5.
PLoS One ; 14(12): e0226302, 2019.
Artigo em Inglês | MEDLINE | ID: mdl-31851695

RESUMO

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.


Assuntos
Astrócitos , Encéfalo/citologia , Separação Celular , Células Endoteliais , Pericitos , Animais , Barreira Hematoencefálica/citologia , Técnicas de Cultura de Células , Feminino , Citometria de Fluxo , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Microscopia Confocal
6.
Sci Transl Med ; 11(518)2019 11 13.
Artigo em Inglês | MEDLINE | ID: mdl-31723036

RESUMO

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.


Assuntos
Molécula de Adesão de Leucócito Ativado/metabolismo , Linfócitos B/citologia , Movimento Celular , Sistema Nervoso Central/metabolismo , Animais , Barreira Hematoencefálica/metabolismo , Encefalomielite Autoimune Experimental/imunologia , Encefalomielite Autoimune Experimental/patologia , Endotélio/metabolismo , Humanos , Memória Imunológica , Camundongos Knockout , Esclerose Múltipla/imunologia , Esclerose Múltipla/patologia , Glicoproteína Mielina-Oligodendrócito/imunologia , Proteínas Recombinantes/imunologia , Índice de Gravidade de Doença
7.
Pharmaceutics ; 11(11)2019 Nov 08.
Artigo em Inglês | MEDLINE | ID: mdl-31717321

RESUMO

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.

8.
Brain ; 142(10): 2979-2995, 2019 10 01.
Artigo em Inglês | MEDLINE | ID: mdl-31412103

RESUMO

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.


Assuntos
Encefalomielite Autoimune Experimental/genética , Encefalomielite Autoimune Experimental/patologia , MicroRNAs/genética , Neurônios/patologia , Animais , Axônios/patologia , Modelos Animais de Doenças , Encefalomielite Autoimune Experimental/metabolismo , Ácido Glutâmico/metabolismo , Humanos , Leucócitos Mononucleares/metabolismo , Camundongos , MicroRNAs/metabolismo , Esclerose Múltipla/genética , Esclerose Múltipla/metabolismo , Esclerose Múltipla/patologia , Degeneração Neural/genética , Degeneração Neural/metabolismo , Degeneração Neural/patologia , Doenças Neurodegenerativas/metabolismo , Neurônios/metabolismo , RNA Mensageiro/metabolismo , Ratos , Ratos Sprague-Dawley , Transdução de Sinais/genética , Medula Espinal/patologia
9.
JCI Insight ; 4(6)2019 03 21.
Artigo em Inglês | MEDLINE | ID: mdl-30895941

RESUMO

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.


Assuntos
Doenças Desmielinizantes/genética , Doenças Desmielinizantes/imunologia , Progressão da Doença , Receptores de Antígenos de Linfócitos T/imunologia , Transferência Adotiva , Animais , Doenças Autoimunes/imunologia , Barreira Hematoencefálica/patologia , Modelos Animais de Doenças , Feminino , Fatores Imunológicos , Masculino , Camundongos , Camundongos Transgênicos , Esclerose Múltipla/imunologia , Fenótipo , Receptores de Antígenos de Linfócitos T/metabolismo , Recidiva , Fatores Sexuais , Linfócitos T/imunologia , Transcriptoma
10.
Sci Rep ; 8(1): 13437, 2018 09 07.
Artigo em Inglês | MEDLINE | ID: mdl-30194392

RESUMO

Multiple sclerosis (MS) is an autoimmune, neurodegenerative disease but the molecular mechanisms underlying neurodegenerative aspects of the disease are poorly understood. microRNAs (miRNAs) are powerful regulators of gene expression that regulate numerous mRNAs simultaneously and can thus regulate programs of gene expression. Here, we describe miRNA expression in neurons captured from mice subjected to experimental autoimmune encephalomyelitis (EAE), a model of central nervous system (CNS) inflammation. Lumbar motor neurons and retinal neurons were laser captured from EAE mice and miRNA expression was assessed by next-generation sequencing and validated by qPCR. We describe 14 miRNAs that are differentially regulated in both neuronal subtypes and determine putative mRNA targets though in silico analysis. Several upregulated neuronal miRNAs are predicted to target pathways that could mediate repair and regeneration during EAE. This work identifies miRNAs that are affected by inflammation and suggests novel candidates that may be targeted to improve neuroprotection in the context of pathological inflammation.


Assuntos
Encefalomielite Autoimune Experimental/metabolismo , Regulação da Expressão Gênica , MicroRNAs/biossíntese , Neurônios Retinianos/metabolismo , Animais , Encefalomielite Autoimune Experimental/genética , Encefalomielite Autoimune Experimental/patologia , Feminino , Inflamação/genética , Inflamação/metabolismo , Inflamação/patologia , Camundongos , MicroRNAs/genética , Neurônios Retinianos/patologia
11.
Proc Natl Acad Sci U S A ; 115(6): E1194-E1203, 2018 02 06.
Artigo em Inglês | MEDLINE | ID: mdl-29358392

RESUMO

Molecular interventions that limit pathogenic CNS inflammation are used to treat autoimmune conditions such as multiple sclerosis (MS). Remarkably, IL-1ß-knockout mice are highly resistant to experimental autoimmune encephalomyelitis (EAE), an animal model of MS. Here, we show that interfering with the IL-1ß/IL-1R1 axis severely impairs the transmigration of myeloid cells across central nervous system (CNS) endothelial cells (ECs). Notably, we report that IL-1ß expression by inflammatory CCR2hi monocytes favors their entry into the spinal cord before EAE onset. Following activation with IL-1ß, CNS ECs release GM-CSF, which in turn converts monocytes into antigen-presenting cells (APCs). Accordingly, spinal cord-infiltrated monocyte-derived APCs are associated with dividing CD4+ T cells. Factors released from the interaction between IL-1ß-competent myeloid cells and CD4+ T cells are highly toxic to neurons. Together, our results suggest that IL-1ß signaling is an entry point for targeting both the initiation and exacerbation of neuroinflammation.


Assuntos
Linfócitos T CD4-Positivos/patologia , Sistema Nervoso Central/patologia , Encefalomielite Autoimune Experimental/patologia , Células Endoteliais/patologia , Interleucina-1beta/fisiologia , Monócitos/patologia , Neurônios/patologia , Animais , Linfócitos T CD4-Positivos/imunologia , Linfócitos T CD4-Positivos/metabolismo , Sistema Nervoso Central/imunologia , Sistema Nervoso Central/metabolismo , Encefalomielite Autoimune Experimental/etiologia , Encefalomielite Autoimune Experimental/metabolismo , Células Endoteliais/imunologia , Células Endoteliais/metabolismo , Fator Estimulador de Colônias de Granulócitos e Macrófagos/metabolismo , Inflamação/etiologia , Inflamação/metabolismo , Inflamação/patologia , Camundongos , Camundongos Knockout , Monócitos/imunologia , Monócitos/metabolismo , Células Mieloides/imunologia , Células Mieloides/metabolismo , Células Mieloides/patologia , Neurônios/imunologia , Neurônios/metabolismo , Receptores CCR2/metabolismo
12.
J Cereb Blood Flow Metab ; 37(8): 2894-2909, 2017 Aug.
Artigo em Inglês | MEDLINE | ID: mdl-28273717

RESUMO

Activated leukocyte cell adhesion molecule (ALCAM) has been proposed to mediate leukocyte migration across the blood-brain barrier (BBB) in multiple sclerosis or experimental autoimmune encephalomyelitis (EAE). Here, we confirmed vascular ALCAM expression in human brain tissue samples in situ and on two different human in vitro BBB models. Antibody-mediated inhibition of ALCAM reduced diapedesis of human CD4+ Th1 but not of Th17 cells across the human BBB in vitro. In accordance to human Th1 cells, mouse Th1 cells showed reduced diapedesis across an ALCAM-/- in vitro BBB model under static but no longer under flow conditions. In contrast to the limited role of ALCAM in T cell extravasation across the BBB, we found a contribution of ALCAM to rolling, adhesion, and diapedesis of human CD14+ monocytes across the human BBB under flow and static conditions. Taken together, our study highlights the potential differences in the CNS expression of ALCAM in mouse and human and supports a prominent role for ALCAM in the multi-step extravasation of monocytes across the BBB.


Assuntos
Antígenos CD/metabolismo , Barreira Hematoencefálica/metabolismo , Moléculas de Adesão Celular Neuronais/metabolismo , Proteínas Fetais/metabolismo , Monócitos/imunologia , Linfócitos T/imunologia , Migração Transendotelial e Transepitelial/imunologia , Animais , Antígenos CD/genética , Barreira Hematoencefálica/imunologia , Moléculas de Adesão Celular Neuronais/genética , Células Cultivadas , Encefalomielite Autoimune Experimental/imunologia , Encefalomielite Autoimune Experimental/metabolismo , Células Endoteliais/imunologia , Células Endoteliais/metabolismo , Endotélio Vascular/metabolismo , Proteínas Fetais/genética , Humanos , Camundongos Endogâmicos C57BL , Camundongos Knockout , Monócitos/metabolismo , Esclerose Múltipla/imunologia , Esclerose Múltipla/metabolismo , Linfócitos T/metabolismo , Migração Transendotelial e Transepitelial/fisiologia
13.
Proc Natl Acad Sci U S A ; 114(4): E524-E533, 2017 01 24.
Artigo em Inglês | MEDLINE | ID: mdl-28069965

RESUMO

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.


Assuntos
Molécula de Adesão de Leucócito Ativado/metabolismo , Encefalomielite Autoimune Experimental/metabolismo , Células Endoteliais/metabolismo , Molécula de Adesão de Leucócito Ativado/genética , Animais , Barreira Hematoencefálica/citologia , Barreira Hematoencefálica/metabolismo , Células Cultivadas , Encefalomielite Autoimune Experimental/patologia , Feminino , Homeostase , Camundongos Endogâmicos C57BL , Camundongos Knockout , Glicoproteína Mielina-Oligodendrócito , Fragmentos de Peptídeos , Índice de Gravidade de Doença , Medula Espinal/metabolismo , Proteínas de Junções Íntimas/metabolismo
14.
Mult Scler ; 23(1): 72-81, 2017 01.
Artigo em Inglês | MEDLINE | ID: mdl-27037182

RESUMO

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.


Assuntos
Linfócitos B/patologia , Esclerose Múltipla/tratamento farmacológico , Esclerose Múltipla/patologia , Natalizumab/uso terapêutico , Síndrome de Abstinência a Substâncias/patologia , Linfócitos T/patologia , Adulto , Linfócitos B/imunologia , Feminino , Humanos , Interferon gama/uso terapêutico , Esclerose Múltipla/diagnóstico por imagem , Linfócitos T/imunologia
15.
J Exp Med ; 213(6): 929-49, 2016 05 30.
Artigo em Inglês | MEDLINE | ID: mdl-27139491

RESUMO

Growing evidence supports a role for IL-1 in multiple sclerosis and experimental autoimmune encephalomyelitis (EAE), but how it impacts neuroinflammation is poorly understood. We show that susceptibility to EAE requires activation of IL-1R1 on radiation-resistant cells via IL-1ß secreted by bone marrow-derived cells. Neutrophils and monocyte-derived macrophages (MDMs) are the main source of IL-1ß and produce this cytokine as a result of their transmigration across the inflamed blood-spinal cord barrier. IL-1R1 expression in the spinal cord is found in endothelial cells (ECs) of the pial venous plexus. Accordingly, leukocyte infiltration at EAE onset is restricted to IL-1R1(+) subpial and subarachnoid vessels. In response to IL-1ß, primary cultures of central nervous system ECs produce GM-CSF, G-CSF, IL-6, Cxcl1, and Cxcl2. Initiation of EAE or subdural injection of IL-1ß induces a similar cytokine/chemokine signature in spinal cord vessels. Furthermore, the transfer of Gr1(+) cells on the spinal cord is sufficient to induce illness in EAE-resistant IL-1ß knockout (KO) mice. Notably, transfer of Gr1(+) cells isolated from C57BL/6 mice induce massive recruitment of recipient myeloid cells compared with cells from IL-1ß KO donors, and this recruitment translates into more severe paralysis. These findings suggest that an IL-1ß-dependent paracrine loop between infiltrated neutrophils/MDMs and ECs drives neuroinflammation.


Assuntos
Encefalomielite Autoimune Experimental/imunologia , Interleucina-1beta/imunologia , Macrófagos/imunologia , Esclerose Múltipla/imunologia , Comunicação Parácrina/imunologia , Medula Espinal/imunologia , Migração Transendotelial e Transepitelial/imunologia , Animais , Encefalomielite Autoimune Experimental/genética , Encefalomielite Autoimune Experimental/patologia , Interleucina-1beta/genética , Macrófagos/patologia , Camundongos , Camundongos Knockout , Esclerose Múltipla/genética , Esclerose Múltipla/patologia , Neutrófilos/imunologia , Neutrófilos/patologia , Comunicação Parácrina/genética , Medula Espinal/patologia , Migração Transendotelial e Transepitelial/genética
16.
Biochim Biophys Acta ; 1862(3): 472-82, 2016 03.
Artigo em Inglês | MEDLINE | ID: mdl-26454208

RESUMO

The blood-brain barrier (BBB) constitutes an elaborate structure formed by specialized capillary endothelial cells, which together with pericytes and perivascular glial cells regulates the exchanges between the central nervous system (CNS) and the periphery. Intricate interactions between the different cellular constituents of the BBB are crucial in establishing a functional BBB and maintaining the delicate homeostasis of the CNS microenvironment. In this review, we discuss the role of astrocytes and microglia in inducing and maintaining barrier properties under physiological conditions as well as their involvement during neuroinflammatory pathologies. This article is part of a Special Issue entitled: Neuro Inflammation edited by Helga E. de Vries and Markus Schwaninger.


Assuntos
Astrócitos/patologia , Barreira Hematoencefálica/patologia , Inflamação/patologia , Leucócitos/patologia , Neuroglia/patologia , Animais , Astrócitos/imunologia , Barreira Hematoencefálica/imunologia , Movimento Celular , Encefalomielite Autoimune Experimental/imunologia , Encefalomielite Autoimune Experimental/patologia , Humanos , Imunidade Celular , Inflamação/imunologia , Leucócitos/imunologia , Microglia/imunologia , Microglia/patologia , Esclerose Múltipla/imunologia , Esclerose Múltipla/patologia , Neuroglia/imunologia , Acoplamento Neurovascular , Pericitos/imunologia , Pericitos/patologia
17.
Ann Neurol ; 78(1): 39-53, 2015 Jul.
Artigo em Inglês | MEDLINE | ID: mdl-25869475

RESUMO

OBJECTIVE: Although Tc17 lymphocytes are enriched in the central nervous system (CNS) of multiple sclerosis (MS) subjects and of experimental autoimmune encephalomyelitis (EAE) animals, limited information is available about their recruitment into the CNS and their role in neuroinflammation. Identification of adhesion molecules used by autoaggressive CD8(+) T lymphocytes to enter the CNS would allow further characterization of this pathogenic subset and could provide new therapeutic targets in MS. We propose that melanoma cell adhesion molecule (MCAM) is a surface marker and adhesion molecule used by pathogenic CD8(+) T lymphocytes to access the CNS. METHODS: Frequency, phenotype, and function of MCAM(+) CD8(+) T lymphocytes was characterized using a combination of ex vivo, in vitro, in situ, and in vivo approaches in humans and mice, including healthy controls, MS subjects, and EAE animals. RESULTS: Herein, we report that MCAM is expressed by human effector CD8(+) T lymphocytes and it is strikingly upregulated during MS relapses. We further demonstrate that MCAM(+) CD8(+) T lymphocytes express more interleukin 17, interferon γ, granulocyte-macrophage colony-stimulating factor, and tumor necrosis factor than MCAM(-) lymphocytes, and exhibit an enhanced killing capacity toward oligodendrocytes. MCAM blockade restricts the transmigration of CD8(+) T lymphocytes across human blood-brain barrier endothelial cells in vitro, and blocking or depleting MCAM in vivo reduces chronic neurological deficits in active, transfer, and spontaneous progressive EAE models. INTERPRETATION: Our data demonstrate that MCAM identifies encephalitogenic CD8(+) T lymphocytes, suggesting that MCAM could represent a biomarker of MS disease activity and a valid target for the treatment of neuroinflammatory conditions.


Assuntos
Barreira Hematoencefálica/metabolismo , Linfócitos T CD8-Positivos/metabolismo , Encefalomielite Autoimune Experimental/metabolismo , Esclerose Múltipla Recidivante-Remitente/metabolismo , Animais , Barreira Hematoencefálica/imunologia , Antígeno CD146/metabolismo , Linfócitos T CD8-Positivos/imunologia , Estudos de Casos e Controles , Encefalomielite Autoimune Experimental/imunologia , Fator Estimulador de Colônias de Granulócitos e Macrófagos/imunologia , Humanos , Técnicas In Vitro , Inflamação , Interferon gama/imunologia , Interleucina-17/imunologia , Camundongos , Camundongos Transgênicos , Esclerose Múltipla Recidivante-Remitente/imunologia , Oligodendroglia , Fator de Necrose Tumoral alfa/imunologia
18.
Brain ; 138(Pt 6): 1598-612, 2015 Jun.
Artigo em Inglês | MEDLINE | ID: mdl-25903786

RESUMO

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.


Assuntos
Barreira Hematoencefálica/metabolismo , Encefalomielite Autoimune Experimental/tratamento farmacológico , Encefalomielite Autoimune Experimental/metabolismo , Inflamação/metabolismo , Esclerose Múltipla/metabolismo , Fatores de Crescimento Neural/fisiologia , Fatores de Crescimento Neural/uso terapêutico , Proteínas Supressoras de Tumor/fisiologia , Proteínas Supressoras de Tumor/uso terapêutico , Animais , Anti-Inflamatórios/farmacologia , Anti-Inflamatórios/uso terapêutico , Proteínas Sanguíneas/metabolismo , Barreira Hematoencefálica/efeitos dos fármacos , Células Endoteliais/metabolismo , Humanos , Inflamação/tratamento farmacológico , Mediadores da Inflamação/metabolismo , Proteínas de Membrana/metabolismo , Camundongos , Camundongos Knockout , Fatores de Crescimento Neural/genética , Fatores de Crescimento Neural/metabolismo , Fatores de Crescimento Neural/farmacologia , Netrina-1 , Permeabilidade , Cultura Primária de Células , Junções Íntimas/metabolismo , Proteínas Supressoras de Tumor/genética , Proteínas Supressoras de Tumor/metabolismo , Proteínas Supressoras de Tumor/farmacologia , Regulação para Cima
19.
J Immunol ; 193(5): 2438-54, 2014 Sep 01.
Artigo em Inglês | MEDLINE | ID: mdl-25049355

RESUMO

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.


Assuntos
Barreira Hematoencefálica/imunologia , Encefalomielite Autoimune Experimental/imunologia , Neutrófilos/imunologia , Medula Espinal/imunologia , Animais , Barreira Hematoencefálica/patologia , Encefalomielite Autoimune Experimental/genética , Encefalomielite Autoimune Experimental/patologia , Feminino , Humanos , Camundongos , Camundongos Transgênicos , Neuromielite Óptica/genética , Neuromielite Óptica/imunologia , Neuromielite Óptica/patologia , Neutrófilos/patologia , Medula Espinal/patologia
20.
PLoS One ; 8(9): e74603, 2013.
Artigo em Inglês | MEDLINE | ID: mdl-24019971

RESUMO

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.


Assuntos
Endotélio Vascular/metabolismo , Proteínas de Fluorescência Verde/genética , Microvasos/metabolismo , Mitocôndrias/metabolismo , Animais , Sequência de Bases , Primers do DNA , Endotélio Vascular/citologia , Citometria de Fluxo , Corantes Fluorescentes , Camundongos , Camundongos Transgênicos , Microvasos/citologia , Reação em Cadeia da Polimerase
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