RESUMO
In this Letter, Dominic Grün and Sagar have been added to the author list (affiliated with Max-Planck-Institute of Immunology and Epigenetics (MPI-IE), Freiburg, Germany). The author list, 'Author contribution' and 'Acknowledgements' sections have been corrected online. See accompanying Amendment.
RESUMO
Microglia have critical roles not only in neural development and homeostasis, but also in neurodegenerative and neuroinflammatory diseases of the central nervous system1-4. These highly diverse and specialized functions may be executed by subsets of microglia that already exist in situ, or by specific subsets of microglia that develop from a homogeneous pool of cells on demand. However, little is known about the presence of spatially and temporally restricted subclasses of microglia in the central nervous system during development or disease. Here we combine massively parallel single-cell analysis, single-molecule fluorescence in situ hybridization, advanced immunohistochemistry and computational modelling to comprehensively characterize subclasses of microglia in multiple regions of the central nervous system during development and disease. Single-cell analysis of tissues of the central nervous system during homeostasis in mice revealed specific time- and region-dependent subtypes of microglia. Demyelinating and neurodegenerative diseases evoked context-dependent subtypes of microglia with distinct molecular hallmarks and diverse cellular kinetics. Corresponding clusters of microglia were also identified in healthy human brains, and the brains of patients with multiple sclerosis. Our data provide insights into the endogenous immune system of the central nervous system during development, homeostasis and disease, and may also provide new targets for the treatment of neurodegenerative and neuroinflammatory pathologies.
Assuntos
Microglia/classificação , Microglia/citologia , Análise de Célula Única , Análise Espaço-Temporal , Animais , Encéfalo/citologia , Encéfalo/patologia , Estudos de Casos e Controles , Separação Celular , Doenças Desmielinizantes/patologia , Feminino , Humanos , Cinética , Masculino , Camundongos , Esclerose Múltipla/patologia , Doenças Neurodegenerativas/patologiaRESUMO
Modeling chronic cortical demyelination allows the study of long-lasting pathological changes observed in multiple sclerosis such as failure of remyelination, chronically disturbed functions of oligodendrocytes, neurons and astrocytes, brain atrophy and cognitive impairments. We aimed at generating an animal model for studying the consequences of chronic cortical demyelination and meningeal inflammation. To induce long-lasting cortical demyelination and chronic meningeal inflammation, we immunized female Lewis rats against myelin oligodendrocyte glycoprotein (MOG) and injected lentiviruses for continuing overexpression of the cytokines TNFα and IFNγ in the cortical brain parenchyma. Immunization with MOG and overexpression of TNFα and IFNγ led to widespread subpial demyelination and meningeal inflammation that were stable for at least 10 weeks. We demonstrate here that immunization with MOG is necessary for acute as well as chronic cortical demyelination. In addition, long-lasting overexpression of TNFα and IFNγ in the brain parenchyma is sufficient to induce chronic meningeal inflammation. Our model simulates key features of chronic cortical demyelination and inflammation, reminiscent of human multiple sclerosis pathology. This will allow molecular, cellular and functional investigations for a better understanding of the adaptation mechanisms of the cerebral cortex in multiple sclerosis.
Assuntos
Esclerose Múltipla , Fator de Necrose Tumoral alfa , Ratos , Animais , Humanos , Feminino , Ratos Endogâmicos Lew , Fator de Necrose Tumoral alfa/genética , Modelos Animais , Glicoproteína Mielina-Oligodendrócito , Córtex Cerebral , InflamaçãoRESUMO
X-linked adrenoleukodystrophy (X-ALD) is the most common leukodystrophy. Despite intensive research in recent years, it remains unclear, what drives the different clinical disease courses. Due to this missing pathophysiological link, therapy for the childhood cerebral disease course of X-ALD (CCALD) remains symptomatic; the allogenic hematopoietic stem cell transplantation or hematopoietic stem-cell gene therapy is an option for early disease stages. The inclusion of dried blood spot (DBS) C26:0-lysophosphatidylcholine to newborn screening in an increasing number of countries is leading to an increasing number of X-ALD patients diagnosed at risk for CCALD. Current follow-up in asymptomatic boys with X-ALD requires repetitive cerebral MRIs under sedation. A reliable and easily accessible biomarker that predicts CCALD would therefore be of great value. Here we report the application of targeted metabolomics by AbsoluteIDQ p180-Kit from Biocrates to search for suitable biomarkers in X-ALD. LysoPC a C20:3 and lysoPC a C20:4 were identified as metabolites that indicate neuroinflammation after induction of experimental autoimmune encephalitis in the serum of Abcd1tm1Kds mice. Analysis of serum from X-ALD patients also revealed different concentrations of these lipids at different disease stages. Further studies in a larger cohort of X-ALD patient sera are needed to prove the diagnostic value of these lipids for use as early biomarkers for neuroinflammation in CCALD patients.
Assuntos
Adrenoleucodistrofia/diagnóstico , Lisofosfatidilcolinas/análise , Metabolômica/métodos , Triagem Neonatal/métodos , Doenças Neuroinflamatórias/etiologia , Membro 1 da Subfamília D de Transportadores de Cassetes de Ligação de ATP/genética , Adrenoleucodistrofia/complicações , Adrenoleucodistrofia/fisiopatologia , Animais , Biomarcadores/sangue , Teste em Amostras de Sangue Seco , Encefalomielite Autoimune Experimental/sangue , Feminino , Humanos , Recém-Nascido , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Doenças Neuroinflamatórias/sangue , FosfolipídeosRESUMO
BACKGROUND: Individuals with impaired immunity are more susceptible to infections than immunocompetent subjects. No vaccines are currently available to induce protection against E. coli meningoencephalitis. This study evaluated the potential of poly(I:C) pre-treatment to induce trained immunity. Poly(I:C) was administered as a non-specific stimulus of innate immune responses to protect immunocompetent and neutropenic wild-type mice from a subsequent challenge by the intracranial injection of E. coli K1. METHODS: Three days prior to infection, mice received an intraperitoneal injection of poly(I:C) or vehicle. Kaplan-Meier survival curves were analyzed. In short-term experiments, bacterial titers and the inflammatory response were characterized in the blood, cerebellum, and spleen homogenates. NK cell subpopulations in the brain and spleen were analyzed by flow cytometry. Numbers of microglia and activation scores were evaluated by histopathology. RESULTS: Pre-treatment with 200 µg poly(I:C) increased survival time, reduced mortality, and enhanced bacterial clearance in the blood, cerebellum, and spleen at early infection in neutropenic mice. Poly(I:C)-mediated protection correlated with an augmented number of NK cells (CD45+NK1.1+CD3-) and Iba-1+ microglial cells and a higher production of IFN-γ in the brain. In the spleen, levels of CCL5/RANTES and IFN-γ were increased and sustained in surviving poly(I:C)-treated animals for 14 days after infection. In immunocompetent animals, survival time was not significantly prolonged in poly(I:C)-treated animals although poly(I:C) priming reduced brain bacterial concentrations compared with vehicle-injected animals at early infection. CONCLUSIONS: Pre-treatment with the viral TLR3 agonist poly(I:C) modulated innate immune responses and strengthened the resistance of neutropenic mice against E. coli K1 meningoencephalitis.
Assuntos
Imunidade Inata/efeitos dos fármacos , Hospedeiro Imunocomprometido/imunologia , Meningite devida a Escherichia coli/imunologia , Poli I-C/farmacologia , Animais , Imunidade Inata/imunologia , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Neutropenia/imunologia , Poli I-C/imunologia , Receptor 3 Toll-Like/agonistas , Receptor 3 Toll-Like/efeitos dos fármacosRESUMO
Proteolipid protein (PLP) is the most abundant integral membrane protein in central nervous system (CNS) myelin. Expression of the Plp-gene in oligodendrocytes is not essential for the biosynthesis of myelin membranes but required to prevent axonal pathology. This raises the question whether the exceptionally high level of PLP in myelin is required later in life, or whether high-level PLP expression becomes dispensable once myelin has been assembled. Both models require a better understanding of the turnover of PLP in myelin in vivo. Thus, we generated and characterized a novel line of tamoxifen-inducible Plp-mutant mice that allowed us to determine the rate of PLP turnover after developmental myelination has been completed, and to assess the possible impact of gradually decreasing amounts of PLP for myelin and axonal integrity. We found that 6 months after targeting the Plp-gene the abundance of PLP in CNS myelin was about halved, probably reflecting that myelin is slowly turned over in the adult brain. Importantly, this reduction by 50% was sufficient to cause the entire spectrum of neuropathological changes previously associated with the developmental lack of PLP, including myelin outfoldings, lamellae splittings, and axonal spheroids. In comparison to axonopathy and gliosis, the infiltration of cytotoxic T-cells was temporally delayed, suggesting a corresponding chronology also in the genetic disorders of PLP-deficiency. High-level abundance of PLP in myelin throughout adult life emerges as a requirement for the preservation of white matter integrity.
Assuntos
Axônios/metabolismo , Sistema Nervoso Central/citologia , Proteína Proteolipídica de Mielina/metabolismo , Bainha de Mielina/metabolismo , Animais , Axônios/ultraestrutura , Citocinas/genética , Citocinas/metabolismo , Antagonistas de Estrogênios/farmacologia , Expressão Gênica/efeitos dos fármacos , Regulação da Expressão Gênica/efeitos dos fármacos , Regulação da Expressão Gênica/genética , Imuno-Histoquímica , Leucócitos Mononucleares/metabolismo , Leucócitos Mononucleares/ultraestrutura , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Transgênicos , Microscopia Eletrônica de Transmissão , Proteínas da Mielina/genética , Proteínas da Mielina/metabolismo , Proteínas da Mielina/ultraestrutura , Proteína Proteolipídica de Mielina/genética , Proteína Proteolipídica de Mielina/ultraestrutura , Bainha de Mielina/ultraestrutura , Proteínas do Tecido Nervoso/genética , Proteínas do Tecido Nervoso/metabolismo , Proteínas do Tecido Nervoso/ultraestrutura , RNA Mensageiro/metabolismo , Tamoxifeno/farmacologiaRESUMO
Optic neuritis is a common manifestation of multiple sclerosis, an inflammatory demyelinating disease of the CNS. Although it is the presenting symptom in many cases, the initial events are currently unknown. However, in the earliest stages of autoimmune optic neuritis in rats, pathological changes are already apparent such as microglial activation and disturbances in myelin ultrastructure of the optic nerves. αB-crystallin is a heat-shock protein induced in cells undergoing cellular stress and has been reported to be up-regulated in both multiple sclerosis and its animal model, experimental autoimmune encephalomyelitis. Therefore, we wished to investigate the timing and localization of its expression in autoimmune optic neuritis. Although loss of oligodendrocytes was not observed until the later disease stages accompanying immune cell infiltration and demyelination, an increase in oligodendrocyte αB-crystallin was observed during the preclinical stages. This was most pronounced within the optic nerve head and was associated with areas of IgG deposition. Since treatment of isolated oligodendrocytes with sera from myelin oligodendrocyte glycoprotein (MOG)-immunized animals induced an increase in αB-crystallin expression, as did passive transfer of sera from MOG-immunized animals to unimmunized recipients, we propose that the partially permeable blood-brain barrier of the optic nerve head may present an opportunity for blood-borne components such as anti-MOG antibodies to come into contact with oligodendrocytes as one of the earliest events in disease development.
Assuntos
Doenças Autoimunes/patologia , Encefalomielite Autoimune Experimental/patologia , Nervo Óptico/patologia , Neurite Óptica/patologia , Animais , Doenças Autoimunes/imunologia , Progressão da Doença , Encefalomielite Autoimune Experimental/imunologia , Feminino , Oligodendroglia/imunologia , Oligodendroglia/patologia , Nervo Óptico/imunologia , Neurite Óptica/imunologia , Ratos , Ratos Sprague-DawleyRESUMO
X-linked adrenoleukodystrophy (X-ALD) and metachromatic leukodystrophy (MLD) are two relatively common examples of hereditary demyelinating diseases caused by a dysfunction of peroxisomal or lysosomal lipid degradation. In both conditions, accumulation of nondegraded lipids leads to the destruction of cerebral white matter. Because of their high lipid content, oligodendrocytes are considered key to the pathophysiology of these leukodystrophies. However, the response to allogeneic stem cell transplantation points to the relevance of cells related to the hematopoietic lineage. In the present study, we aimed to better characterize the pathogenetic role of microglia in the above-mentioned diseases. Applying recently established microglia markers to human autopsy cases of X-ALD and MLD we were able to delineate distinct lesion stages in evolving demyelinating lesions. The immune-phenotype of microglia was altered already early in lesion evolution, and microglia loss preceded full-blown myelin degeneration both in X-ALD and MLD. DNA fragmentation indicating phagocyte death was observed in areas showing microglia loss. The morphology and dynamics of phagocyte decay differed between the diseases and between lesion stages, hinting at distinct pathways of programmed cell death. In summary, the present study shows an early and severe damage to microglia in the pathogenesis of X-ALD and MLD. This hints at a central pathophysiologic role of these cells in the diseases and provides evidence for an ongoing transfer of toxic substrates primarily enriched in myelinating cells to microglia.
Assuntos
Adrenoleucodistrofia/patologia , Leucodistrofia Metacromática/patologia , Microglia/patologia , Bainha de Mielina/patologia , Adolescente , Adrenoleucodistrofia/genética , Adrenoleucodistrofia/metabolismo , Adulto , Idoso , Criança , Pré-Escolar , Estudos de Coortes , Feminino , Humanos , Leucodistrofia Metacromática/genética , Leucodistrofia Metacromática/metabolismo , Masculino , Microglia/metabolismo , Pessoa de Meia-Idade , Bainha de Mielina/genética , Bainha de Mielina/metabolismoRESUMO
BACKGROUND: Quinoline-3-carboxamides, such as laquinimod, ameliorate CNS autoimmunity in patients and reduce tumor cell metastasis experimentally. Previous studies have focused on the immunomodulatory effect of laquinimod on myeloid cells. The data contained herein suggest that quinoline-3-carboxamides improve the immunomodulatory and anti-tumor effects of NK cells by upregulating the adhesion molecule DNAX accessory molecule-1 (DNAM-1). METHODS: We explored how NK cell activation by laquinimod inhibits CNS autoimmunity in experimental autoimmune encephalomyelitis (EAE), the most utilized model of MS, and improves immunosurveillance of experimental lung melanoma metastasis. Functional manipulations included in vivo NK and DC depletion experiments and in vitro assays of NK cell function. Clinical, histological, and flow cytometric read-outs were assessed. RESULTS: We demonstrate that laquinimod activates natural killer (NK) cells via the aryl hydrocarbon receptor and increases their DNAM-1 cell surface expression. This activation improves the cytotoxicity of NK cells against B16F10 melanoma cells and augments their immunoregulatory functions in EAE by interacting with CD155+ dendritic cells (DC). Noteworthy, the immunosuppressive effect of laquinimod-activated NK cells was due to decreasing MHC class II antigen presentation by DC and not by increasing DC killing. CONCLUSIONS: This study clarifies how DNAM-1 modifies the bidirectional crosstalk of NK cells with CD155+ DC, which can be exploited to suppress CNS autoimmunity and strengthen tumor surveillance.
Assuntos
Antígenos de Diferenciação de Linfócitos T/metabolismo , Autoimunidade/efeitos dos fármacos , Células Dendríticas/efeitos dos fármacos , Vigilância Imunológica/imunologia , Células Matadoras Naturais/efeitos dos fármacos , Quinolonas/farmacologia , Animais , Antígenos de Diferenciação de Linfócitos T/imunologia , Autoimunidade/imunologia , Células Dendríticas/imunologia , Encefalomielite Autoimune Experimental/imunologia , Humanos , Células Matadoras Naturais/imunologia , Ativação Linfocitária/efeitos dos fármacos , Melanoma Experimental , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Transgênicos , Quinolinas/agonistas , Receptores de Hidrocarboneto Arílico/agonistas , Receptores Virais/imunologiaRESUMO
BACKGROUND: The adaptive immune system has been considered to play a minimal role in the early host response during bacterial meningitis. METHODS: We investigated the progression and outcome of pneumococcal meningitis in Rag1-/- mice lacking functional B and T cells by assessing overall and symptom-free survival, bacteriological and histological studies, as well as flow cytometry and measurements of proinflammatory mediators. RESULTS: The intracerebral injection of S. pneumoniae D39 induced the recruitment of B and T cells (CD4+, γδ and natural killer) into the brain of wild-type mice. Mice with no functional B and T cells developed clinical symptoms and succumbed to the infection earlier than the wild-type group. In the CNS, Rag1-/- mice showed lower levels of interleukin 1ß, reduced microglial proliferation, and impaired granulocyte recruitment with an earlier spread of pneumococci into the bloodstream, compared with wild-type mice. Lack of B and T cells resulted in a severe impairment of bacterial clearance in blood, spleen, and liver and an exaggerated systemic inflammatory response. CONCLUSIONS: B and T cells are important effector cells delaying the spread of pneumococci from the brain to the systemic circulation and shaping the immune response, thereby prolonging the survival of the host in the absence of antibiotic treatment.
Assuntos
Imunidade Adaptativa , Encéfalo/imunologia , Meningite Pneumocócica/imunologia , Meningite Pneumocócica/fisiopatologia , Streptococcus pneumoniae/imunologia , Animais , Linfócitos B/imunologia , Encéfalo/microbiologia , Encéfalo/ultraestrutura , Citocinas/biossíntese , Interleucina-1beta/imunologia , Células Matadoras Naturais , Meningite Pneumocócica/microbiologia , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Knockout , Microglia/imunologia , Baço/microbiologia , Linfócitos T/imunologiaRESUMO
Cortical demyelination is a widely recognized hallmark of multiple sclerosis (MS) and correlate of disease progression and cognitive decline. The pathomechanisms initiating and driving gray matter damage are only incompletely understood. Here, we determined the infiltrating leukocyte subpopulations in 26 cortical demyelinated lesions of biopsied MS patients and assessed their contribution to cortical lesion formation in a newly developed mouse model. We find that conformation-specific anti-myelin antibodies contribute to cortical demyelination even in the absence of the classical complement pathway. T cells and natural killer cells are relevant for intracortical type 2 but dispensable for subpial type 3 lesions, whereas CCR2+ monocytes are required for both. Depleting CCR2+ monocytes in marmoset monkeys with experimental autoimmune encephalomyelitis using a novel humanized CCR2 targeting antibody translates into significantly less cortical demyelination and disease severity. We conclude that biologics depleting CCR2+ monocytes might be attractive candidates for preventing cortical lesion formation and ameliorating disease progression in MS.
Assuntos
Córtex Cerebral/imunologia , Encefalomielite Autoimune Experimental/imunologia , Monócitos/imunologia , Esclerose Múltipla/imunologia , Adulto , Animais , Callithrix , Córtex Cerebral/patologia , Estudos de Coortes , Encefalomielite Autoimune Experimental/patologia , Feminino , Humanos , Células Matadoras Naturais/imunologia , Células Matadoras Naturais/patologia , Masculino , Meninges/imunologia , Meninges/patologia , Camundongos Endogâmicos C57BL , Camundongos Transgênicos , Pessoa de Meia-Idade , Monócitos/patologia , Esclerose Múltipla/patologia , Distribuição Aleatória , Receptores CCR2/metabolismo , Linfócitos T/imunologia , Linfócitos T/patologiaRESUMO
BACKGROUND: Meningoencephalitis caused by Escherichia coli is associated with high rates of mortality and risk of neurological sequelae in newborns and infants and in older or immunocompromised adults. A high prevalence of neurological disorders has been observed in geriatric populations at risk of hypovitaminosis D. METHODS: In vivo, we studied the effects of vitamin D3 on survival and the host's immune response in experimental bacterial meningoencephalitis in mice after intracerebral E. coli infection. To produce different systemic vitamin D3 concentrations, mice received a low, standard, or high dietary vitamin D3 supplementation. Bacterial titers in blood, spleen, and brain homogenates were determined. Leukocyte infiltration was assessed by histological scores, and tissue cytokine or chemokine concentrations were measured. RESULTS: Mice fed a diet with low vitamin D3 concentration died earlier than control animals after intracerebral infection. Vitamin D deficiency did not inhibit leukocyte recruitment into the subarachnoid space and did not lead to an increased density of bacteria in blood, spleen, or brain homogenates. The release of proinflammatory interleukin (IL)-6 was decreased and the release of anti-inflammatory IL-10 was increased in mice fed a diet with high vitamin D3 supplementation. CONCLUSION: Our observations suggest a detrimental role of vitamin D deficiency in bacterial central nervous system infections. Vitamin D may exert immune regulatory functions.
Assuntos
Colecalciferol/deficiência , Infecções por Escherichia coli/complicações , Escherichia coli/patogenicidade , Meningoencefalite/etiologia , Meningoencefalite/mortalidade , Deficiência de Vitamina D , Análise de Variância , Animais , Carga Bacteriana/métodos , Peso Corporal , Sistema Nervoso Central/metabolismo , Sistema Nervoso Central/microbiologia , Sistema Nervoso Central/patologia , Colecalciferol/administração & dosagem , Colecalciferol/sangue , Citocinas/metabolismo , Suplementos Nutricionais , Modelos Animais de Doenças , Regulação da Expressão Gênica/efeitos dos fármacos , Meningoencefalite/patologia , Camundongos , Camundongos Endogâmicos C57BL , Baço/metabolismo , Baço/microbiologia , Baço/patologia , Fatores de TempoRESUMO
AIMS: Natalizumab is a humanized monoclonal antibody specific for CD49d receptors of integrins. It inhibits the entry of inflammatory cells into the central nervous system and is approved for the treatment of relapsing-remitting multiple sclerosis (MS). Several lines of evidence indicate an involvement of B cells and plasma cells in MS pathogenesis. However, treatment with the natalizumab analogon PS/2 immunoglobulin G (IgG) has so far only been investigated in T cell-mediated animal models of MS. Due to the importance of B lineage cells in the pathogenesis of MS, the objective of the present study has thus been to analyse the effects of PS/2 IgG in a mouse model of MS with T and B cell cooperation (OSE mice). METHODS: OSE mice were treated with the natalizumab analogon PS/2 IgG either at disease onset or after peak of disease. Treatment was also performed with PS/2 F(ab')2 fragments. RESULTS: PS/2 IgG treatment improved the clinical outcome and decreased spinal cord demyelination and immune cell infiltration if given early in the disease course. Treatment increased blood leukocytes and resulted in a partial internalization of CD49d in T and B cells. The therapeutic effects of PS/2 IgG injections were independent of the Fc fragment as F(ab')2 injections were equally beneficial. In contrast, PS/2 IgG was not effective when given late in the disease course. CONCLUSIONS: Results indicate that natalizumab may also be beneficial in MS with B cell-driven immunopathogenesis.
Assuntos
Linfócitos B/imunologia , Esclerose Múltipla/tratamento farmacológico , Esclerose Múltipla/patologia , Natalizumab/administração & dosagem , Animais , Sítios de Ligação , Modelos Animais de Doenças , Imunoglobulina G/administração & dosagem , Integrina alfa4/imunologia , Leucócitos/efeitos dos fármacos , Leucócitos/metabolismo , Camundongos , Camundongos Transgênicos , Esclerose Múltipla/imunologia , Medula Espinal/efeitos dos fármacos , Medula Espinal/patologia , Substância Branca/efeitos dos fármacos , Substância Branca/patologiaRESUMO
BACKGROUND: Prophylaxis with unmethylated cytosine phosphate guanidine (CpG) oligodeoxynucleotides (ODN) protects against several systemic experimental infections. Escherichia coli is a major cause of Gram-negative neonatal bacterial meningitis and also causes meningitis and meningoencephalitis in older and immunocompromised patients. METHODS: Wild-type (wt) and Toll-like receptor 9 (TLR9)-deficient mice were rendered neutropenic by intraperitoneal administration of the anti-Ly-6G monoclonal antibody. Immunocompetent and neutropenic mice received intraperitoneal CpG ODN or vehicle 72 h prior to induction of E. coli K1 meningoencephalitis. RESULTS: Pre-treatment with CpG ODN significantly increased survival of neutropenic wt mice from 33% to 75% (P = 0.0003) but did not protect neutropenic TLR9-/- mice. The protective effect of CpG ODN was associated with an enhanced production of interleukin (IL)-12/IL-23p40 with sustained increased levels in serum and spleen at least for 17 days after conditioning compared to buffer-treated animals. CpG-treated neutropenic wt mice showed reduced bacterial concentrations and increased recruitment of Ly6ChighCCR2+ monocytes in brain and spleen 42 h after infection. The levels of macrophage inflammatory protein 1α (MIP-1α) and interferon gamma (IFN-γ) in spleen were higher 42 h after infection in CpG-treated compared to buffer-treated neutropenic animals. In immunocompetent mice, prophylaxis with CpG ODN did not significantly increase survival compared to the buffer group (60% vs. 45%, P = 0.2). CONCLUSIONS: These findings suggest that systemic administration of CpG ODN may help to prevent bacterial CNS infections in immunocompromised individuals.
Assuntos
Infecções por Escherichia coli/prevenção & controle , Guanidina/química , Oligodesoxirribonucleotídeos/uso terapêutico , Animais , Antígenos CD/metabolismo , Sistema Nervoso Central/efeitos dos fármacos , Sistema Nervoso Central/microbiologia , Sistema Nervoso Central/patologia , Citocinas/metabolismo , Modelos Animais de Doenças , Esquema de Medicação , Escherichia coli/fisiologia , Citometria de Fluxo , Meningoencefalite/prevenção & controle , Camundongos , Camundongos Knockout , Baço/microbiologia , Baço/patologia , Receptor Toll-Like 9/deficiênciaRESUMO
Neuromyelitis optica (NMO) is a chronic, mostly relapsing inflammatory demyelinating disease of the CNS characterized by serum anti-aquaporin 4 (AQP4) antibodies in the majority of patients. Anti-AQP4 antibodies derived from NMO patients target and deplete astrocytes in experimental models when co-injected with complement. However, the time course and mechanisms of oligodendrocyte loss and demyelination and the fate of oligodendrocyte precursor cells (OPC) have not been examined in detail. Also, no studies regarding astrocyte repopulation of experimental NMO lesions have been reported. We utilized two rat models using either systemic transfer or focal intracerebral injection of recombinant human anti-AQP4 antibodies to generate NMO-like lesions. Time-course experiments were performed to examine oligodendroglial and astroglial damage and repair. In addition, oligodendrocyte pathology was studied in early human NMO lesions. Apart from early complement-mediated astrocyte destruction, we observed a prominent, very early loss of oligodendrocytes and oligodendrocyte precursor cells (OPCs) as well as a delayed loss of myelin. Astrocyte repopulation of focal NMO lesions was already substantial after 1 week. Olig2-positive OPCs reappeared before NogoA-positive, mature oligodendrocytes. Thus, using two experimental models that closely mimic the human disease, our study demonstrates that oligodendrocyte and OPC loss is an extremely early feature in the formation of human and experimental NMO lesions and leads to subsequent, delayed demyelination, highlighting an important difference in the pathogenesis of MS and NMO.
Assuntos
Encéfalo/patologia , Neuromielite Óptica/patologia , Oligodendroglia/patologia , Medula Espinal/patologia , Adulto , Idoso , Animais , Aquaporina 4/metabolismo , Fatores de Transcrição Hélice-Alça-Hélice Básicos/metabolismo , Citocinas/metabolismo , Modelos Animais de Doenças , Feminino , Humanos , Masculino , Pessoa de Meia-Idade , Proteína Básica da Mielina/metabolismo , Proteínas da Mielina/metabolismo , Proteínas do Tecido Nervoso/metabolismo , Proteínas Nogo , Fator de Transcrição 2 de Oligodendrócitos , Oligodendroglia/metabolismo , Ratos Endogâmicos LewRESUMO
OBJECTIVES: To assess neurofilament light chain serum (sNfL) levels in patients with secondary progressive multiple sclerosis (SP-MS). METHODS: Using a single molecule array, we analyzed sNfL levels in a cross-sectional cohort study of 153 patients with SP-MS hospitalized for rehabilitation in a clinic specialized in the care for patients with multiple sclerosis (MS). In addition, we investigated the correlation of disease activity with sNfL levels in 36 patients with relapsing-remitting MS (RR-MS). RESULTS: Mean sNfL levels in patients with SP-MS were consistently elevated when compared with age-matched controls and patients with RR-MS. In SP-MS, age dependency of sNfL levels was pronounced, whereas patients with RR-MS younger than 41 years without recent disease activity were not distinguishable from age-matched healthy controls. In a multivariate analysis, clinical disability was a risk factor for elevated sNfL levels in SP-MS, whereas no correlation with comorbidities, such as cardiovascular disease, diabetes mellitus, smoking status, or vitamin D serum levels, could be detected. DISCUSSION: These findings highlight that measurement of sNfL levels represents a useful tool to assess the extent of neuroaxonal damage as a surrogate for clinical progression in patients with SP-MS, when age and disease activity as major confounders are taken into account.
Assuntos
Esclerose Múltipla Crônica Progressiva , Esclerose Múltipla Recidivante-Remitente , Proteínas de Neurofilamentos , Humanos , Estudos Transversais , Masculino , Feminino , Pessoa de Meia-Idade , Esclerose Múltipla Crônica Progressiva/sangue , Esclerose Múltipla Crônica Progressiva/fisiopatologia , Adulto , Proteínas de Neurofilamentos/sangue , Esclerose Múltipla Recidivante-Remitente/sangue , Esclerose Múltipla Recidivante-Remitente/fisiopatologia , Idoso , Fatores Etários , Estudos de Coortes , Avaliação da Deficiência , Biomarcadores/sangueRESUMO
Magnetic resonance imaging (MRI) is the gold standard for the detection of multiple sclerosis (MS) lesions. However, current MRI techniques provide little information about the structural features of a brain lesion with inflammatory cell infiltration, demyelination, gliosis, acute axonal damage and axonal loss. To identify methods for a differentiation of demyelination, inflammation, and axonal damage we developed a novel mouse model combining cuprizone-induced demyelination and experimental autoimmune encephalomyelitis. MS-like brain lesions were assessed by T1-weighted, T2-weighted, and magnetization transfer MRI as well as by diffusion tensor imaging (DTI). T2-weighted MRI differentiated control and diseased mice, while T1-weighted MRI better reflected the extent of inflammation and axonal damage. In DTI, axonal damage and cellular infiltration led to a reduction of the axial diffusivity, whereas primary demyelination after cuprizone treatment was reflected by changes in radial but not axial diffusivity. Importantly, alterations in radial diffusivity were less pronounced in mice with demyelination, inflammation, and acute axonal damage, indicating that radial diffusivity may underestimate demyelination in acute MS lesions. In conclusion, the combined information from different DTI parameters allows for a more precise identification of solely demyelinated lesions versus demyelinated and acutely inflamed lesions. These findings are of relevance for offering individualized, stage-adapted therapies for MS patients.
Assuntos
Encéfalo/patologia , Imagem de Tensor de Difusão/métodos , Encefalomielite Autoimune Experimental/patologia , Imageamento por Ressonância Magnética/métodos , Esclerose Múltipla/patologia , Animais , Axônios/patologia , Barreira Hematoencefálica/patologia , Cuprizona , Doenças Desmielinizantes/patologia , Análise Discriminante , Encefalomielite Autoimune Experimental/induzido quimicamente , Feminino , Gliose/patologia , Processamento de Imagem Assistida por Computador/métodos , Inflamação/patologia , Camundongos , Camundongos Endogâmicos C57BL , Inibidores da Monoaminoxidase , Esclerose Múltipla/induzido quimicamente , Toxina Pertussis/toxicidadeRESUMO
Active immunization with amyloid-ß (Aß) peptide 1-42 reverses amyloid plaque deposition in the CNS of patients with Alzheimer's disease and in amyloid precursor protein transgenic mice. However, this treatment may also cause severe, life-threatening meningoencephalitis. Physiological responses to immunization with Aß(1-42) are poorly understood. In this study, we characterized cognitive and immunological consequences of Aß(1-42)/CFA immunization in C57BL/6 mice. In contrast to mice immunized with myelin oligodendrocyte glycoprotein (MOG)(35-55)/CFA or CFA alone, Aß(1-42)/CFA immunization resulted in impaired exploratory activity, habituation learning, and spatial-learning abilities in the open field. As morphological substrate of this neurocognitive phenotype, we identified a disseminated, nonfocal immune cell infiltrate in the CNS of Aß(1-42)/CFA-immunized animals. In contrast to MOG(35-55)/CFA and PBS/CFA controls, the majority of infiltrating cells in Aß(1-42)/CFA-immunized mice were CD11b(+)CD14(+) and CD45(high), indicating their blood-borne monocyte/macrophage origin. Immunization with Aß(1-42)/CFA was significantly more potent than immunization with MOG(35-55)/CFA or CFA alone in activating macrophages in the secondary lymphoid compartment and peripheral tissues. Studies with TLR2/4-deficient mice revealed that the TLR2/4 pathway mediated the Aß(1-42)-dependent proinflammatory cytokine release from cells of the innate immune system. In line with this, TLR2/4 knockout mice were protected from cognitive impairment upon immunization with Aß(1-42)/CFA. Thus, this study identifies adjuvant effects of Aß(1-42), which result in a clinically relevant neurocognitive phenotype highlighting potential risks of Aß immunotherapy.
Assuntos
Vacinas contra Alzheimer/efeitos adversos , Peptídeos beta-Amiloides/imunologia , Imunidade Inata/imunologia , Transtornos da Memória/imunologia , Fragmentos de Peptídeos/imunologia , Vacinação/efeitos adversos , Adjuvantes Imunológicos/farmacologia , Vacinas contra Alzheimer/imunologia , Peptídeos beta-Amiloides/farmacologia , Animais , Comportamento Animal/efeitos dos fármacos , Encéfalo/imunologia , Encéfalo/patologia , Separação Celular , Citocinas/biossíntese , Ensaio de Imunoadsorção Enzimática , Feminino , Citometria de Fluxo , Adjuvante de Freund/imunologia , Adjuvante de Freund/farmacologia , Humanos , Macrófagos/imunologia , Transtornos da Memória/patologia , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Knockout , Monócitos/imunologia , Fragmentos de Peptídeos/farmacologia , Reação em Cadeia da Polimerase Via Transcriptase Reversa , Receptor 2 Toll-Like/biossíntese , Receptor 2 Toll-Like/imunologia , Receptor 4 Toll-Like/biossíntese , Receptor 4 Toll-Like/imunologiaRESUMO
Experimental autoimmune encephalomyelitis (EAE) is a relevant animal model for the human demyelinating inflammatory disorder of the central nervous system (CNS), multiple sclerosis (MS). Induction of EAE by adoptive transfer allows studying the role of the donor T lymphocyte in disease pathogenesis. It has been challenging to reliably induce adoptive transfer EAE in C57BL/6 (H-2b) mice. The goal of this study was to develop a reproducible and high yield protocol for adoptive transfer EAE in C57BL/6 mice. A step-wise experimental approach permitted us to develop a protocol that resulted in a consistent relatively high disease incidence of ~70% in recipient mice. Donor mice were immunized with myelin oligodendrocyte glycoprotein (MOG)p35-55 in complete Freund's adjuvant (CFA) followed by pertussis toxin (PT). Only lymph node cells (LNC) isolated at day 12 post immunization, and restimulated in vitro for 72 hours with 10 µg/mL of MOGp35-55 and 0.5 ng/mL of interleukin-12 (IL-12) were able to transfer disease. The ability of LNC to transfer disease was associated with the presence of inflammatory infiltrates in the CNS at day 12. Interferon gamma (IFNγ) was produced at comparable levels in cell cultures prepared from mice at both day 6 and day 12 post immunization. By contrast, there was a trend towards a negative association between IL-17 and disease susceptibility in our EAE model. The amount of GM-CSF secreted was significantly increased in the culture supernatants from cells collected at day 12 post immunization versus those collected at day 6 post-immunization. Activated CD4+ T cells present in the day 12 LNC cultures maintained expression of the transcription factor T-bet, which has been shown to regulate the expression of the IL-23 receptor. Also, there was an increased prevalence of MOGp35-55-specific CD4+ T cells in day 12 LNC after in vitro re-stimulation. In summary, encephalitogenic LNC that adoptively transfer EAE in C57BL/6 mice were not characterized by a single biomarker in our study, but by a composite of inflammatory markers. Our data further suggest that GM-CSF expression by CD4+ T cells regulated by IL-23 contributes to their encephalitogenicity in our EAE model.
Assuntos
Transferência Adotiva/métodos , Linfócitos T CD4-Positivos/fisiologia , Encefalomielite Autoimune Experimental/imunologia , Fator Estimulador de Colônias de Granulócitos e Macrófagos/imunologia , Linfonodos/citologia , Proteínas com Domínio T/imunologia , Animais , Biomarcadores/metabolismo , Encéfalo/imunologia , Encéfalo/patologia , Linfócitos T CD4-Positivos/citologia , Linfócitos T CD4-Positivos/imunologia , Encefalomielite Autoimune Experimental/fisiopatologia , Glicoproteínas/administração & dosagem , Glicoproteínas/imunologia , Humanos , Interferon gama/imunologia , Interleucina-12/imunologia , Interleucina-17/imunologia , Camundongos , Camundongos Endogâmicos C57BL , Glicoproteína Mielina-Oligodendrócito , Fragmentos de Peptídeos/administração & dosagem , Fragmentos de Peptídeos/imunologia , Medula Espinal/imunologia , Medula Espinal/patologiaRESUMO
The primary biological function of the endogenous cellular prion protein has remained unclear. We investigated its biological function in the generation of cellular immune responses using cellular prion protein gene-specific small interfering ribonucleic acid in vivo and in vitro. Our results were confirmed by blocking cellular prion protein with monovalent antibodies and by using cellular prion protein-deficient and -transgenic mice. In vivo prion protein gene-small interfering ribonucleic acid treatment effects were of limited duration, restricted to secondary lymphoid organs and resulted in a 70% reduction of cellular prion protein expression in leukocytes. Disruption of cellular prion protein signalling augmented antigen-specific activation and proliferation, and enhanced T cell receptor signalling, resulting in zeta-chain-associated protein-70 phosphorylation and nuclear factor of activated T cells/activator protein 1 transcriptional activity. In vivo prion protein gene-small interfering ribonucleic acid treatment promoted T cell differentiation towards pro-inflammatory phenotypes and increased survival of antigen-specific T cells. Cellular prion protein silencing with small interfering ribonucleic acid also resulted in the worsening of actively induced and adoptively transferred experimental autoimmune encephalomyelitis. Finally, treatment of myelin basic protein(1-11) T cell receptor transgenic mice with prion protein gene-small interfering ribonucleic acid resulted in spontaneous experimental autoimmune encephalomyelitis. Thus, central nervous system autoimmune disease was modulated at all stages of disease: the generation of the T cell effector response, the elicitation of T effector function and the perpetuation of cellular immune responses. Our findings indicate that cellular prion protein regulates T cell receptor-mediated T cell activation, differentiation and survival. Defects in autoimmunity are restricted to the immune system and not the central nervous system. Our data identify cellular prion protein as a regulator of cellular immunological homoeostasis and suggest cellular prion protein as a novel potential target for therapeutic immunomodulation.