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1.
Artigo em Inglês | MEDLINE | ID: mdl-36446612

RESUMO

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.


Assuntos
Encefalite , Encefalomielite Autoimune Experimental , Esclerose Múltipla , Doenças Neurodegenerativas , Humanos , Animais , Camundongos , Encefalomielite Autoimune Experimental/genética , Transcriptoma , Esclerose Múltipla/genética , Encéfalo , Endotélio
2.
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
3.
Artigo em Inglês | MEDLINE | ID: mdl-36241608

RESUMO

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.


Assuntos
Esclerose Múltipla , Humanos , Encéfalo/metabolismo , Proteína de Membrana Semelhante a Receptor de Coxsackie e Adenovirus/metabolismo , Células Endoteliais/metabolismo , Leucócitos/metabolismo , Leucócitos Mononucleares , Fator de Necrose Tumoral alfa/metabolismo
4.
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
5.
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
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.
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
8.
Cell Mol Immunol ; 16(7): 652-665, 2019 07.
Artigo em Inglês | MEDLINE | ID: mdl-30635649

RESUMO

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.


Assuntos
Ligante CD27/metabolismo , Sistema Nervoso Central/imunologia , Encefalomielite Autoimune Experimental/imunologia , Esclerose Múltipla/imunologia , Células Th1/imunologia , Células Th17/imunologia , Transferência Adotiva , Animais , Células Cultivadas , Humanos , Inflamação , Ativação Linfocitária , Camundongos , Camundongos Endogâmicos C57BL , Transdução de Sinais , Membro 7 da Superfamília de Receptores de Fatores de Necrose Tumoral/metabolismo
9.
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
10.
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
11.
Neurobiol Dis ; 74: 14-24, 2015 Feb.
Artigo em Inglês | MEDLINE | ID: mdl-25448765

RESUMO

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.


Assuntos
Barreira Hematoencefálica/metabolismo , Encefalomielite Autoimune Experimental/metabolismo , Esclerose Múltipla Crônica Progressiva/metabolismo , Esclerose Múltipla Recidivante-Remitente/metabolismo , Adulto , Idoso , Animais , Astrócitos/metabolismo , Astrócitos/patologia , Barreira Hematoencefálica/patologia , Encéfalo/metabolismo , Encéfalo/patologia , Encefalomielite Autoimune Experimental/patologia , Endotélio Vascular/metabolismo , Endotélio Vascular/patologia , Feminino , Gliose/metabolismo , Gliose/patologia , Humanos , Estudos Longitudinais , Masculino , Camundongos Transgênicos , Pessoa de Meia-Idade , Esclerose Múltipla Crônica Progressiva/patologia , Esclerose Múltipla Recidivante-Remitente/patologia , Linfócitos T/metabolismo
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