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1.
EMBO J ; 35(1): 89-101, 2016 Jan 04.
Artigo em Inglês | MEDLINE | ID: mdl-26612827

RESUMO

Multiple sclerosis is the most frequent chronic inflammatory disease of the CNS. The entry and survival of pathogenic T cells in the CNS are crucial for the initiation and persistence of autoimmune neuroinflammation. In this respect, contradictory evidence exists on the role of the most potent type of antigen-presenting cells, dendritic cells. Applying intravital two-photon microscopy, we demonstrate the gatekeeper function of CNS professional antigen-presenting CD11c(+) cells, which preferentially interact with Th17 cells. IL-17 expression correlates with expression of GM-CSF by T cells and with accumulation of CNS CD11c(+) cells. These CD11c(+) cells are organized in perivascular clusters, targeted by T cells, and strongly express the inflammatory chemokines Ccl5, Cxcl9, and Cxcl10. Our findings demonstrate a fundamental role of CNS CD11c(+) cells in the attraction of pathogenic T cells into and their survival within the CNS. Depletion of CD11c(+) cells markedly reduced disease severity due to impaired enrichment of pathogenic T cells within the CNS.


Assuntos
Células Apresentadoras de Antígenos/fisiologia , Encéfalo/patologia , Antígeno CD11c/análise , Células Dendríticas/fisiologia , Encefalomielite Autoimune Experimental/patologia , Linfócitos T/imunologia , Animais , Células Apresentadoras de Antígenos/química , Encéfalo/imunologia , Movimento Celular , Células Dendríticas/química , Encefalomielite Autoimune Experimental/imunologia , Fator Estimulador de Colônias de Granulócitos e Macrófagos/metabolismo , Interleucina-17/metabolismo , Camundongos Endogâmicos C57BL , Linfócitos T/fisiologia , Células Th17/fisiologia
2.
J Immunol ; 200(8): 2554-2562, 2018 04 15.
Artigo em Inglês | MEDLINE | ID: mdl-29549177

RESUMO

Multiple sclerosis (MS) is the most common chronic inflammatory demyelinating disease of the CNS. Myelin-specific CD4+ Th lymphocytes are known to play a major role in both MS and its animal model experimental autoimmune encephalomyelitis (EAE). CCR7 is a critical element for immune cell trafficking and recirculation, that is, lymph node homing, under homeostatic conditions; blocking CCR7+ central memory cells from egress of lymph nodes is a therapeutic approach in MS. To define the effect of CD4+ T cell-specific constitutive deletion of CCR7 in the priming and effector phase in EAE, we used an active EAE approach in T cell reconstituted Rag1-/- mice, as well as adoptive transfer EAE, in which mice received in vitro-primed CCR7-/- or CCR7+/+ myelin Ag TCR-transgenic 2d2 Th17 cells. Two-photon laser scanning microscopy was applied in living anesthetized mice to monitor the trafficking of CCR7-deficient and wild-type CD4+ T cells in inflammatory lesions within the CNS. We demonstrate that CD4+ T cell-specific constitutive deletion of CCR7 led to impaired induction of active EAE. In adoptive transfer EAE, mice receiving in vitro-primed CCR7-/- 2d2 Th17 cells showed similar disease onset as mice adoptively transferred with CCR7+/+ 2d2 Th17 cells. Using two-photon laser scanning microscopy CCR7-/- and CCR7+/+ CD4+ T cells did not reveal differences in motility in either animal model of MS. These findings indicate a crucial role of CCR7 in neuroinflammation during the priming of autoimmune CD4+ T cells but not in the CNS.


Assuntos
Linfócitos T CD4-Positivos/imunologia , Encefalomielite Autoimune Experimental/imunologia , Receptores CCR7/imunologia , Animais , Sistema Nervoso Central/imunologia , Modelos Animais de Doenças , Linfonodos/imunologia , Camundongos , Camundongos Endogâmicos C57BL , Esclerose Múltipla/imunologia , Receptores de Antígenos de Linfócitos T/imunologia , Células Th17/imunologia
3.
Mult Scler ; 23(4): 567-576, 2017 Apr.
Artigo em Inglês | MEDLINE | ID: mdl-27436357

RESUMO

OBJECTIVE: Natalizumab is known to prevent T-helper cells entering the central nervous system (CNS). We hypothesize that more pathogenic T-helper cells are present outside the CNS and a possible relationship to disease severity. METHODS: Characterization and enrichment of human CD4+IL-17+ cells were performed ex vivo using peripheral blood mononuclear cells from natalizumab-treated relapsing-remitting multiple sclerosis (RRMS) patients ( n = 33), untreated RRMS patients ( n = 13), and healthy controls ( n = 33). Magnetic resonance imaging (MRI) scans were performed routinely for patients. RESULTS: Lymphocytes were elevated in peripheral blood of natalizumab-treated patients compared to untreated patients and healthy controls. Whereas group comparison for CD4+IL-17+ numbers also differed, CD4+IFN-γ+ and CD4+IL-22+ counts were not increased. CD4+IL-17+ cells not only expressed but also secreted IL-17. In natalizumab-treated patients, IL-17+ cell frequency was found to correlate with T1-hypointense lesions, but was not an indicator for rebound activity after treatment discontinuation, except in one patient who experienced a fulminant rebound, and interestingly, in whom the highest IL-17+ cell levels were observed. CONCLUSION: Increased lymphocytes and CD4+IL-17+ cells in the blood of RRMS patients receiving natalizumab corroborate the drug's mechanism of action, that is, blocking transmigration to CNS. Correlation between IL-17-expressing lymphocytes and T1-hypointense lesions underlines the important role of these cells in the disease pathology.


Assuntos
Interleucina-17/metabolismo , Leucócitos Mononucleares/efeitos dos fármacos , Esclerose Múltipla/tratamento farmacológico , Natalizumab/uso terapêutico , Adolescente , Adulto , Linfócitos T CD4-Positivos/efeitos dos fármacos , Sistema Nervoso Central/efeitos dos fármacos , Sistema Nervoso Central/patologia , Feminino , Humanos , Masculino , Pessoa de Meia-Idade , Adulto Jovem
4.
J Neurosci ; 35(12): 4837-50, 2015 Mar 25.
Artigo em Inglês | MEDLINE | ID: mdl-25810515

RESUMO

Multiple sclerosis (MS) is an inflammatory disease of the CNS thought to be driven by CNS-specific T lymphocytes. Although CD8(+) T cells are frequently found in multiple sclerosis lesions, their distinct role remains controversial because direct signs of cytotoxicity have not been confirmed in vivo. In the present work, we determined that murine ovalbumin-transgenic (OT-1) CD8(+) T cells recognize the myelin peptide myelin oligodendrocyte glycoprotein 40-54 (MOG40-54) both in vitro and in vivo. The aim of this study was to investigate whether such cross-recognizing CD8(+) T cells are capable of inducing CNS damage in vivo. Using intravital two-photon microscopy in the mouse model of multiple sclerosis, we detected antigen recognition motility of the OT-1 CD8(+) T cells within the CNS leading to a selective enrichment in inflammatory lesions. However, this cross-reactivity of OT-1 CD8(+) T cells with MOG peptide in the CNS did not result in clinically or subclinically significant damage, which is different from myelin-specific CD4(+) Th17-mediated autoimmune pathology. Therefore, intravital imaging demonstrates that local myelin recognition by autoreactive CD8(+) T cells in inflammatory CNS lesions alone is not sufficient to induce disability or increase axonal injury.


Assuntos
Linfócitos T CD4-Positivos/imunologia , Linfócitos T CD8-Positivos/imunologia , Sistema Nervoso Central/imunologia , Encefalomielite Autoimune Experimental/patologia , Esclerose Múltipla/patologia , Glicoproteína Mielina-Oligodendrócito/imunologia , Degeneração Neural/imunologia , Animais , Autoimunidade/imunologia , Morte Celular , Proliferação de Células , Células Cultivadas , Sistema Nervoso Central/patologia , Encefalomielite Autoimune Experimental/imunologia , Feminino , Masculino , Camundongos , Camundongos Transgênicos , Esclerose Múltipla/imunologia
5.
J Neurochem ; 136(5): 971-80, 2016 Mar.
Artigo em Inglês | MEDLINE | ID: mdl-26662167

RESUMO

In multiple sclerosis (MS), a candidate downstream mechanism for neuronal injury is glutamate (Glu)-induced excitotoxicity, leading to toxic increases in intraneuronal Ca(2+) . Here, we used in vivo two-photon imaging in the brain of TN-XXL transgenic Ca(2+) reporter mice to test whether promising oral MS therapeutics, namely fingolimod, dimethyl fumarate, and their respective metabolites fingolimod-phosphate and monomethyl fumarate, can protect neurons against acute glutamatergic excitotoxic damage. We also assessed whether these drugs can protect against excitotoxicity in vitro using primary cortical neurons, and whether they can directly inhibit Glu release from pathogenic T-helper 17 lymphocytes. In vivo, direct and acute (1 h) administration of 100 mM Glu to the brainstem resulted in a rapid and significant up-regulation in neuronal Ca(2+) signaling as well as morphological excitotoxic changes that were attenuated by the NMDA-receptor antagonist MK801. Direct CNS administration of MS drugs prior to Glu significantly delayed or reduced, but did not prevent the neuronal Ca(2+) increase or morphological changes. In vitro, prolonged (24 h) treatment of primary neurons with the fumarates significantly protected against neurotoxicity induced by Glu as well as NMDA, similar to MK801. Furthermore, monomethyl fumerate significantly reduced Glu release from pathogenic T-helper 17 lymphocytes. Overall, these data suggest that MS drugs may mediate neuroprotection via excitotoxicity modulating effects. Evidence suggests MS pathogenesis may involve neuronal excitotoxicity, induced by local release of glutamate. However, current MS drugs, including dimethyl fumerate (DMF) and fingolimod (FTY720) are largely anti-inflammatory and not yet fully tested for their neuroprotective potential. Here, we show that the drugs, in particular DMF metabolite monomethyl fumerate (MMF), protect neurons by excitotoxicity modulating effects. Th17, T-helper 17.


Assuntos
Morte Celular/efeitos dos fármacos , Ácido Glutâmico/metabolismo , Imunomodulação/imunologia , Esclerose Múltipla/tratamento farmacológico , Neurônios/metabolismo , Fármacos Neuroprotetores/farmacologia , Animais , Encéfalo/efeitos dos fármacos , Encéfalo/metabolismo , Células Cultivadas , Ácido Caínico/farmacologia , Esclerose Múltipla/imunologia , Esclerose Múltipla/metabolismo , Transdução de Sinais/efeitos dos fármacos , Regulação para Cima
6.
Acta Neuropathol ; 129(2): 279-95, 2015 02.
Artigo em Inglês | MEDLINE | ID: mdl-25500713

RESUMO

The contribution of microglia to ischemic cortical stroke is of particular therapeutic interest because of the impact on the survival of brain tissue in the ischemic penumbra, a region that is potentially salvable upon a brain infarct. Whether or not tissue in the penumbra survives critically depends on blood flow and vessel perfusion. To study the role of microglia in cortical stroke and blood vessel stability, CX3CR1(+/GFP) mice were subjected to transient middle cerebral artery occlusion and then microglia were investigated using time-lapse two-photon microscopy in vivo. Soon after reperfusion, microglia became activated in the stroke penumbra and started to expand cellular protrusions towards adjacent blood vessels. All microglia in the penumbra were found associated with blood vessels within 24 h post reperfusion and partially fully engulfed them. In the same time frame blood vessels became permissive for blood serum components. Migration assays in vitro showed that blood serum proteins leaking into the tissue provided molecular cues leading to the recruitment of microglia to blood vessels and to their activation. Subsequently, these perivascular microglia started to eat up endothelial cells by phagocytosis, which caused an activation of the local endothelium and contributed to the disintegration of blood vessels with an eventual break down of the blood brain barrier. Loss-of-microglia-function studies using CX3CR1(GFP/GFP) mice displayed a decrease in stroke size and a reduction in the extravasation of contrast agent into the brain penumbra as measured by MRI. Potentially, medication directed at inhibiting microglia activation within the first day after stroke could stabilize blood vessels in the penumbra, increase blood flow, and serve as a valuable treatment for patients suffering from ischemic stroke.


Assuntos
Isquemia Encefálica/fisiopatologia , Encéfalo/irrigação sanguínea , Encéfalo/fisiopatologia , Microglia/fisiologia , Acidente Vascular Cerebral/fisiopatologia , Animais , Barreira Hematoencefálica/patologia , Barreira Hematoencefálica/fisiopatologia , Encéfalo/patologia , Isquemia Encefálica/patologia , Receptor 1 de Quimiocina CX3C , Linhagem Celular , Modelos Animais de Doenças , Proteínas de Fluorescência Verde/genética , Proteínas de Fluorescência Verde/metabolismo , Camundongos da Linhagem 129 , Camundongos Endogâmicos C57BL , Camundongos Transgênicos , Microglia/patologia , Fagocitose/fisiologia , Receptores de Quimiocinas/genética , Receptores de Quimiocinas/metabolismo , Acidente Vascular Cerebral/patologia
7.
Exp Parasitol ; 129(3): 270-6, 2011 Nov.
Artigo em Inglês | MEDLINE | ID: mdl-21819984

RESUMO

Leishmania (L.) major is a protozoan parasite that infects mammalian hosts and causes a spectrum of disease manifestations that is strongly associated with the genetic background of the host. Interleukin (IL)-6 is an acute phase proinflammatory cytokine, known in vitro to be involved in the inhibition of the generation of regulatory T cells. IL-6-deficient mice were infected with L. major, and T cell and monocyte subsets were analyzed with flow cytometry. Our data show that at the site of infection in the footpad and in the draining popliteal lymph node, numbers of regulatory T cells remain unchanged between WT and IL-6-deficient mice. However, the spleens of IL-6(-/-) mice contained fewer regulatory T cells after infection with L. major. The development of cutaneous lesions is similar between WT and IL-6-deficient mice, while parasite burden in IL-6(-/-) mice is reduced compared to WT. The development of IFN-γ or IL-10 producing T cells is similar in IL-6(-/-) mice. Despite a comparable adaptive T cell response, IL-6-deficient mice develop an earlier peak of some inflammatory cytokines than WT mice. This data indicate that the role of IL-6 in the differentiation of regulatory T cells is complex in vivo, and the effect of an absence of this cytokine can be counter-intuitive.


Assuntos
Interleucina-6/imunologia , Leishmania major/imunologia , Leishmaniose Cutânea/imunologia , Monócitos/citologia , Subpopulações de Linfócitos T/citologia , Animais , Citocinas/sangue , Modelos Animais de Doenças , Feminino , Masculino , Camundongos , Camundongos Endogâmicos BALB C , Camundongos Endogâmicos C57BL , Monócitos/classificação , Monócitos/imunologia , Óxido Nítrico Sintase Tipo II/biossíntese , Organismos Livres de Patógenos Específicos , Subpopulações de Linfócitos T/imunologia
8.
Front Neurol ; 10: 205, 2019.
Artigo em Inglês | MEDLINE | ID: mdl-30915022

RESUMO

Multiple sclerosis (MS) is a chronic autoimmune disease of the central nervous system (CNS) leading to CNS inflammation and neurodegeneration. Current anti-inflammatory drugs have only limited efficacy on progressive neurodegenerative processes underlining the need to understand immune-mediated neuronal injury. Cell adhesion molecules play an important role for immune cell migration over the blood-brain barrier whereas their role in mediating potentially harmful contacts between invading immune cells and neurons is incompletely understood. Here, we assess the role of the CNS-specific neuronal adhesion molecule ICAM-5 using experimental autoimmune encephalomyelitis (EAE), an animal model of MS. ICAM-5 knockout mice show a more severe EAE disease course in the chronic phase indicating a neuroprotective function of ICAM-5 in progressive neurodegeneration. In agreement with the predominant CNS-specific function of ICAM-5, lymphocyte function-associated antigen 1 (LFA-1)/ICAM-1 contact between antigen-presenting cells and T helper (Th)17 cells in EAE is not affected by ICAM-5. Strikingly, intrathecal application of the shed soluble form, sICAM-5, ameliorates EAE disease symptoms and thus might serve locally as an endogenous neuronal defense mechanism which is activated upon neuroinflammation in the CNS. In humans, cerebrospinal fluid from patients suffering from progressive forms of MS shows decreased sICAM-5 levels, suggesting a lack of this endogenous protective pathway in these patient groups. Overall, our study points toward a novel role of ICAM-5 in CNS autoinflammation in progressive EAE/MS.

9.
Cell Stress Chaperones ; 23(1): 77-88, 2018 01.
Artigo em Inglês | MEDLINE | ID: mdl-28687980

RESUMO

Necrosis is a form of cell death that is detrimental to the affected tissue because the cell ruptures and releases its content (reactive oxygen species among others) into the extracellular space. Clusterin (CLU), a cytoprotective extracellular chaperone has been shown to be upregulated in the face of necrosis. We here show that in addition to CLU upregulation, necrotic cell lysates induce JNK/SAPK signaling, the IRE1α branch of the unfolded protein response (UPR), the MAPK/ERK1/2, and the mTOR signaling pathways and results in an enhanced proliferation of the vital surrounding cells. We name this novel response mechanism: Necrosis-induced Proliferation (NiP).


Assuntos
Endorribonucleases/metabolismo , Proteínas Serina-Treonina Quinases/metabolismo , Resposta a Proteínas não Dobradas , Proliferação de Células , Sobrevivência Celular , Clusterina/metabolismo , Células HEK293 , Resposta ao Choque Térmico , Humanos , Proteínas Quinases JNK Ativadas por Mitógeno/metabolismo , NF-kappa B/metabolismo , Necrose , Fosforilação , Transdução de Sinais , Fator de Transcrição AP-1/metabolismo , Proteína 1 de Ligação a X-Box/metabolismo
10.
J Neurosci Methods ; 249: 8-15, 2015 Jul 15.
Artigo em Inglês | MEDLINE | ID: mdl-25864804

RESUMO

BACKGROUND: Irreversible axonal and neuronal damage are the correlate of disability in patients suffering from multiple sclerosis (MS). A sustained increase of cytoplasmic free [Ca(2+)] is a common upstream event of many neuronal and axonal damage processes and could represent an early and potentially reversible step. NEW METHOD: We propose a method to specifically analyze the neurodegenerative aspects of experimental autoimmune encephalomyelitis by Förster Resonance Energy Transfer (FRET) imaging of neuronal and axonal Ca(2+) dynamics by two-photon laser scanning microscopy (TPLSM). RESULTS: Using the genetically encoded Ca(2+) sensor TN-XXL expressed in neurons and their corresponding axons, we confirm the increase of cytoplasmic free [Ca(2+)] in axons and neurons of autoimmune inflammatory lesions compared to those in non-inflamed brains. We show that these relative [Ca(2+)] increases were associated with immune-neuronal interactions. COMPARISON WITH EXISTING METHODS: In contrast to Ca(2+)-sensitive dyes the use of a genetically encoded Ca(2+) sensor allows reliable intraaxonal free [Ca(2+)] measurements in living anesthetized mice in health and disease. This method detects early axonal damage processes in contrast to e.g. cell/axon morphology analysis, that rather detects late signs of neurodegeneration. CONCLUSIONS: Thus, we describe a method to analyze and monitor early neuronal damage processes in the brain in vivo.


Assuntos
Tronco Encefálico/patologia , Cálcio , Encefalomielite Autoimune Experimental/patologia , Transferência Ressonante de Energia de Fluorescência/métodos , Neurônios/patologia , Animais , Axônios/patologia , Camundongos , Microscopia Confocal
11.
PLoS One ; 8(9): e75303, 2013.
Artigo em Inglês | MEDLINE | ID: mdl-24073260

RESUMO

Clusterin, also known as apolipoprotein J, is expressed from a variety of tissues and implicated in pathological disorders such as neurodegenerative diseases, ischemia and cancer. In contrast to secretory clusterin (sCLU), which acts as an extracellular chaperone, the synthesis, subcellular localization and function(s) of intracellular CLU isoforms is currently a matter of intense discussion. By investigating human CLU mRNAs we here unravel mechanisms leading to the synthesis of distinct CLU protein isoforms and analyze their subcellular localization and their impact on apoptosis and on NF-κB-activity. Quantitative PCR-analyses revealed the expression of four different stress-inducible CLU mRNA variants in non-cancer and cancer cell lines. In all cell lines variant 1 represents the most abundant mRNA, whereas all other variants collectively account for no more than 0.34% of total CLU mRNA, even under stressed conditions. Overexpression of CLU cDNAs combined with in vitro mutagenesis revealed distinct translational start sites including a so far uncharacterized non-canonical CUG start codon. We show that all exon 2-containing mRNAs encode sCLU and at least three non-glycosylated intracellular isoforms, CLU1­449, CLU21­449 and CLU34­449, which all reside in the cytosol of unstressed and stressed HEK­293 cells. The latter is the only form expressed from an alternatively spliced mRNA variant lacking exon 2. Functional analysis revealed that none of these cytosolic CLU forms modulate caspase-mediated intrinsic apoptosis or significantly affects TNF-α-induced NF-κB-activity. Therefore our data challenge some of the current ideas regarding the physiological functions of CLU isoforms in pathologies.


Assuntos
Apoptose , Clusterina/metabolismo , NF-kappa B/metabolismo , Processamento de Proteína Pós-Traducional , RNA Mensageiro/genética , Proteína X Associada a bcl-2/metabolismo , Western Blotting , Células CACO-2 , Proliferação de Células , Clusterina/genética , Células HEK293 , Humanos , Técnicas Imunoenzimáticas , Luciferases/metabolismo , Células MCF-7 , Dados de Sequência Molecular , NF-kappa B/genética , Isoformas de Proteínas , Reação em Cadeia da Polimerase em Tempo Real , Reação em Cadeia da Polimerase Via Transcriptase Reversa , Transdução de Sinais , Proteína X Associada a bcl-2/genética
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