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BACKGROUND & AIMS: Although T-cell intrinsic expression of G9a has been associated with murine intestinal inflammation, mechanistic insight into the role of this methyltransferase in human T-cell differentiation is ill defined, and manipulation of G9a function for therapeutic use against inflammatory disorders is unexplored. METHODS: Human naive T cells were isolated from peripheral blood and differentiated in vitro in the presence of a G9a inhibitor (UNC0642) before being characterized via the transcriptome (RNA sequencing), chromatin accessibility (assay for transposase-accessible chromatin by sequencing), protein expression (cytometry by time of flight, flow cytometry), metabolism (mitochondrial stress test, ultrahigh performance liquid chromatography-tandem mas spectroscopy) and function (T-cell suppression assay). The in vivo role of G9a was assessed using 3 murine models. RESULTS: We discovered that pharmacologic inhibition of G9a enzymatic function in human CD4 T cells led to spontaneous generation of FOXP3+ T cells (G9a-inibitors-T regulatory cells [Tregs]) in vitro that faithfully reproduce human Tregs, functionally and phenotypically. Mechanistically, G9a inhibition altered the transcriptional regulation of genes involved in lipid biosynthesis in T cells, resulting in increased intracellular cholesterol. Metabolomic profiling of G9a-inibitors-Tregs confirmed elevated lipid pathways that support Treg development through oxidative phosphorylation and enhanced lipid membrane composition. Pharmacologic G9a inhibition promoted Treg expansion in vivo upon antigen (gliadin) stimulation and ameliorated acute trinitrobenzene sulfonic acid-induced colitis secondary to tissue-specific Treg development. Finally, Tregs lacking G9a expression (G9a-knockout Tregs) remain functional chronically and can rescue T-cell transfer-induced colitis. CONCLUSION: G9a inhibition promotes cholesterol metabolism in T cells, favoring a metabolic profile that facilitates Treg development in vitro and in vivo. Our data support the potential use of G9a inhibitors in the treatment of immune-mediated conditions including inflammatory bowel disease.
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Linfocitos T CD4-Positivos , Colitis , Ratones , Humanos , Animales , Metabolismo de los Lípidos , Linfocitos T Reguladores/metabolismo , Colitis/inducido químicamente , Colitis/tratamiento farmacológico , Colitis/genética , Cromatina , Inflamación , Colesterol , Lípidos , Factores de Transcripción Forkhead/metabolismoRESUMEN
BACKGROUND AND OBJECTIVES: We sought to determine clinical significance of neuronal septin autoimmunity and evaluate for potential IgG effects. METHODS: Septin-IgGs were detected by indirect immunofluorescence assays (IFAs; mouse tissue and cell based) or Western blot. IgG binding to (and internalization of) extracellular septin epitopes were evaluated for by live rat hippocampal neuron assay. The impact of purified patient IgGs on murine cortical neuron function was determined by recording extracellular field potentials in a multielectrode array platform. RESULTS: Septin-IgGs were identified in 23 patients. All 8 patients with septin-5-IgG detected had cerebellar ataxia, and 7 had prominent eye movement disorders. One of 2 patients with co-existing septin-7-IgG had additional psychiatric phenotype (apathy, emotional blunting, and poor insight). Fifteen patients had septin-7 autoimmunity, without septin-5-IgG detected. Disorders included encephalopathy (11; 2 patients with accompanying myelopathy, and 2 were relapsing), myelopathy (3), and episodic ataxia (1). Psychiatric symptoms (≥1 of agitation, apathy, catatonia, disorganized thinking, and paranoia) were prominent in 6 of 11 patients with encephalopathic symptoms. Eight of 10 patients with data available (from 23 total) improved after immunotherapy, and a further 2 patients improved spontaneously. Staining of plasma membranes of live hippocampal neurons produced by patient IgGs (subclasses 1 and 2) colocalized with pre- and post-synaptic markers. Decreased spiking and bursting behavior in mixed cultures of murine glutamatergic and GABAergic cortical neurons produced by patient IgGs were attributable to neither antigenic crosslinking and internalization nor complement activation. INTERPRETATION: Septin-IgGs are predictive of distinct treatment-responsive autoimmune central nervous system (CNS) disorders. Live neuron binding and induced electrophysiologic effects by patient IgGs may support septin-specific pathophysiology. ANN NEUROL 2022;92:1090-1101.
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Encefalopatías , Enfermedades de la Médula Espinal , Animales , Ratas , Ratones , Septinas/metabolismo , Autoinmunidad , Neuronas/metabolismo , Inmunoglobulina G/metabolismoRESUMEN
New onset refractory status epilepticus (NORSE), including its subtype with a preceding febrile illness known as febrile infection-related epilepsy syndrome (FIRES), is one of the most severe forms of status epilepticus. The exact causes of NORSE are currently unknown, and there is so far no disease-specific therapy. Identifying the underlying pathophysiology and discovering specific biomarkers, whether immunologic, infectious, genetic, or other, may help physicians in the management of patients with NORSE. A broad spectrum of biomarkers has been proposed for status epilepticus patients, some of which were evaluated for patients with NORSE. Nonetheless, none has been validated, due to significant variabilities in study cohorts, collected biospecimens, applied analytical methods, and defined outcome endpoints, and to small sample sizes. The NORSE Institute established an open NORSE/FIRES biorepository for health-related data and biological samples allowing the collection of biospecimens worldwide, promoting multicenter research and sharing of data and specimens. Here, we suggest standard operating procedures for biospecimen collection and biobanking in this rare condition. We also propose criteria for the appropriate use of previously collected biospecimens. We predict that the widespread use of standardized procedures will reduce heterogeneity, facilitate the future identification of validated biomarkers for NORSE, and provide a better understanding of the pathophysiology and best clinical management for these patients.
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Epilepsia Refractaria , Encefalitis , Estado Epiléptico , Humanos , Bancos de Muestras Biológicas , Estado Epiléptico/tratamiento farmacológico , Convulsiones/complicaciones , Epilepsia Refractaria/terapia , Encefalitis/complicaciones , BiomarcadoresRESUMEN
Neuromyelitis optica is an autoimmune inflammatory disorder targeting aquaporin-4 water channels in CNS astrocytes. Histopathological descriptions of astrocytic lesions reported in neuromyelitis optica so far have emphasized a characteristic loss of aquaporin-4, with deposition of IgG and complement and lysis of astrocytes, but sublytic reactions have been underappreciated. We performed a multi-modality study of 23 neuromyelitis optica autopsy cases (clinically and/or pathologically confirmed; 337 tissue blocks). By evaluating astrocytic morphology, immunohistochemistry and AQP4 RNA transcripts, and their associations with demyelinating activity, we documented a spectrum of astrocytopathy in addition to complement deposition, microglial reaction, granulocyte infiltration and regenerating activity. Within advanced demyelinating lesions, and in periplaque areas, there was remarkable hypertrophic astrogliosis, more subtle than astrocytic lysis. A degenerative component was suggested by 'dystrophic' morphology, cytoplasmic vacuolation, Rosenthal fibres and associated stress protein markers. The abundance of AQP4 mRNA transcripts in sublytic reactive astrocytes devoid of aquaporin-4 protein supported in vivo restoration following IgG-induced aquaporin-4 endocytosis/degradation. Astrocytic alterations extending beyond demyelinating lesions speak to astrocytopathy being an early and primary event in the evolving neuromyelitis optica lesion. Focal astrocytopathy observed without aquaporin-4 loss or lytic complement component deposition verifies that astrocytic reactions in neuromyelitis optica are not solely dependent on IgG-mediated aquaporin-4 loss or lysis by complement or by IgG-dependent leucocyte mediators. We conclude that neuromyelitis optica reflects a global astrocytopathy, initiated by binding of IgG to aquaporin-4 and not simply definable by demyelination and astrocytic lysis. The spectrum of astrocytic morphological changes in neuromyelitis optica attests to the complexity of factors influencing the range of astrocytic physiological responses to a targeted attack by aquaporin-4-specific IgG. Sublytic astrocytic reactions are no doubt an important determinant of the lesion's evolution and potential for repair. Pharmacological manipulation of the astrocytic stress response may offer new avenues for therapeutic intervention.
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Neuromielitis Óptica , Acuaporina 4 , Astrocitos/metabolismo , Humanos , Inmunoglobulina G/metabolismo , Neuromielitis Óptica/metabolismoRESUMEN
The causes of grey matter pathology and diffuse neuron injury in MS remain incompletely understood. Axonal stress signals arising from white matter lesions has been suggested to play a role in initiating this diffuse grey matter pathology. Therefore, to identify the most upstream transcriptional responses in neurons arising from demyelinated axons, we analyzed the transcriptome of actively translating neuronal transcripts in mouse models of demyelinating disease. Among the most upregulated genes, we identified transcripts associated with the ISGylation pathway. ISGylation refers to the covalent attachment of the ubiquitin-like molecule interferon stimulated gene (ISG) 15 to lysine residues on substrates targeted by E1 ISG15-activating enzyme, E2 ISG15-conjugating enzymes and E3 ISG15-protein ligases. We further confirmed that ISG15 expression is increased in MS cortical and deep gray matter. Upon investigating the functional impact of neuronal ISG15 upregulation, we noted that ISG15 expression was associated changes in neuronal extracellular vesicle protein and miRNA cargo. Specifically, extracellular vesicle-associated miRNAs were skewed toward increased frequency of proinflammatory and neurotoxic miRNAs and decreased frequency of anti-inflammatory and neuroprotective miRNAs. Furthermore, we found that ISG15 directly activated microglia in a CD11b-dependent manner and that microglial activation was potentiated by treatment with EVs from neurons expressing ISG15. Further study of the role of ISG15 and ISGylation in neurons in MS and neurodegenerative diseases is warranted.
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Enfermedades Desmielinizantes , MicroARNs , Ratones , Animales , Ubiquitinas/genética , Ubiquitinas/química , Ubiquitinas/metabolismo , Microglía/metabolismo , Citocinas/genética , Citocinas/metabolismo , Lisina , Interferones , Ubiquitina-Proteína Ligasas/metabolismo , Neuronas/metabolismoRESUMEN
BACKGROUND: The pathogenic contribution of neuroinflammation to ictogenesis and epilepsy may provide a therapeutic target for reduction of seizure burden in patients that are currently underserved by traditional anti-seizure medications. The Theiler's murine encephalomyelitis virus (TMEV) model has provided important insights into the role of inflammation in ictogenesis, but questions remain regarding the relative contribution of microglia and inflammatory monocytes in this model. METHODS: Female C57BL/6 mice were inoculated by intracranial injection of 2 × 105, 5 × 104, 1.25 × 104, or 3.125 × 103 plaque-forming units (PFU) of the Daniel's strain of TMEV at 4-6 weeks of age. Infiltration of inflammatory monocytes, microglial activation, and cytokine production were measured at 24 h post-infection (hpi). Viral load, hippocampal injury, cognitive performance, and seizure burden were assessed at several timepoints. RESULTS: The intensity of inflammatory infiltration and the extent of hippocampal injury induced during TMEV encephalitis scaled with the amount of infectious virus in the initial inoculum. Cognitive performance was preserved in mice inoculated with 1.25 × 104 PFU TMEV relative to 2 × 105 PFU TMEV, but peak viral load at 72 hpi was equivalent between the inocula. CCL2 production in the brain was attenuated by 90% and TNFα and IL6 production was absent in mice inoculated with 1.25 × 104 PFU TMEV. Acute infiltration of inflammatory monocytes was attenuated by more than 80% in mice inoculated with 1.25 × 104 PFU TMEV relative to 2 × 105 PFU TMEV but microglial activation was equivalent between groups. Seizure burden was attenuated and the threshold to kainic acid-induced seizures was higher in mice inoculated with 1.25 × 104 PFU TMEV but low-level behavioral seizures persisted and the EEG exhibited reduced but detectable abnormalities. CONCLUSIONS: The size of the inflammatory monocyte response induced by TMEV scales with the amount of infectious virus in the initial inoculum, despite the development of equivalent peak infectious viral load. In contrast, the microglial response does not scale with the inoculum, as microglial hyper-ramification and increased Iba-1 expression were evident in mice inoculated with either 1.25 × 104 or 2 × 105 PFU TMEV. Inoculation conditions that drive inflammatory monocyte infiltration resulted in robust behavioral seizures and EEG abnormalities, but the low inoculum condition, associated with only microglial activation, drove a more subtle seizure and EEG phenotype.
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Microglía , Theilovirus , Animales , Femenino , Humanos , Ratones , Ratones Endogámicos C57BL , Microglía/metabolismo , Monocitos/metabolismo , Convulsiones/patologíaRESUMEN
BACKGROUND: Microglia are the primary phagocytes of the central nervous system and are responsible for removing damaged myelin following demyelination. Previous investigations exploring the consequences of myelin phagocytosis on microglial activation overlooked the biochemical modifications present on myelin debris. Such modifications, including citrullination, are increased within the inflammatory environment of multiple sclerosis lesions. METHODS: Mouse cortical myelin isolated by ultracentrifugation was citrullinated ex vivo by incubation with the calcium-dependent peptidyl arginine deiminase PAD2. Demyelination was induced by 6 weeks of cuprizone (0.3%) treatment and spontaneous repair was initiated by reversion to normal chow. Citrullinated or unmodified myelin was injected into the primary motor cortex above the cingulum bundle at the time of reversion to normal chow and the consequent impact on remyelination was assessed by measuring the surface area of myelin basic protein-positive fibers in the cortex 3 weeks later. Microglial responses to myelin were characterized by measuring cytokine release, assessing flow cytometric markers of microglial activation, and RNAseq profiling of transcriptional changes. RESULTS: Citrullinated myelin induced a unique microglial response marked by increased tumor necrosis factor α (TNFα) production both in vitro and in vivo. This response was not induced by unmodified myelin. Injection of citrullinated myelin but not unmodified myelin into the cortex of cuprizone-demyelinated mice significantly inhibited spontaneous remyelination. Antibody-mediated neutralization of TNFα blocked this effect and restored remyelination to normal levels. CONCLUSIONS: These findings highlight the role of post-translation modifications such as citrullination in the determination of microglial activation in response to myelin during demyelination. The inhibition of endogenous repair induced by citrullinated myelin and the reversal of this effect by neutralization of TNFα may have implications for therapeutic approaches to patients with inflammatory demyelinating disorders.
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Quelantes , Citrulina/química , Cuprizona , Enfermedades Desmielinizantes/inducido químicamente , Enfermedades Desmielinizantes/patología , Microglía/metabolismo , Vaina de Mielina/química , Factor de Necrosis Tumoral alfa/biosíntesis , Animales , Células Cultivadas , Citocinas/metabolismo , Ratones , Ratones Endogámicos C57BL , Microglía/efectos de los fármacos , Microinyecciones , Corteza Motora , Proteína Básica de MielinaRESUMEN
OBJECTIVE: We recently reported successful treatment of a child with febrile infection-related epilepsy syndrome (FIRES), a subtype of new onset refractory status epilepticus, with the recombinant interleukin-1 (IL1) receptor antagonist (IL1RA) anakinra. On this basis, we tested whether endogenous IL1RA production or function is deficient in FIRES patients. METHODS: Levels of IL1ß and IL1RA were measured in serum and cerebrospinal fluid (CSF). The inhibitory activity of endogenous IL1RA was assessed using a cell-based reporter assay. IL1RN gene variants were identified by sequencing. Expression levels for the secreted and intracellular isoforms of IL1RA were measured in patient and control cells by real-time polymerase chain reaction. RESULTS: Levels of endogenous IL1RA and IL1ß were elevated in the serum and CSF of patients with FIRES (n = 7) relative to healthy controls (n = 10). Serum from FIRES patients drove IL1R signaling activity and potentiated IL1R signaling in response to exogenous IL1ß in a cell-based reporter assay. Functional assessment of endogenous IL1RA activity in 3 FIRES patients revealed attenuated inhibition of IL1R signaling. Sequencing of IL1RN in our index patient revealed multiple variants. This was accompanied by reduced expression of intracellular but not secreted isoforms of IL1RA in the patient's peripheral blood mononuclear cells. INTERPRETATION: Our findings suggest that FIRES is associated with reduced expression of intracellular IL1RA isoforms and a functional deficiency in IL1RA inhibitory activity. These observations may provide insight into disease pathogenesis for FIRES and other inflammatory seizure disorders and may provide a valuable biomarker for therapeutic decision-making. Ann Neurol 2019;85:526-537.
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Epilepsia Refractaria/metabolismo , Síndromes Epilépticos/metabolismo , Infecciones/metabolismo , Proteína Antagonista del Receptor de Interleucina 1/sangre , Proteína Antagonista del Receptor de Interleucina 1/líquido cefalorraquídeo , Convulsiones Febriles/metabolismo , Biomarcadores/sangre , Biomarcadores/líquido cefalorraquídeo , Epilepsia Refractaria/diagnóstico , Epilepsia Refractaria/tratamiento farmacológico , Síndromes Epilépticos/diagnóstico , Síndromes Epilépticos/tratamiento farmacológico , Femenino , Células HEK293 , Humanos , Infecciones/diagnóstico , Infecciones/tratamiento farmacológico , Proteína Antagonista del Receptor de Interleucina 1/uso terapéutico , Masculino , Convulsiones Febriles/diagnóstico , Convulsiones Febriles/tratamiento farmacológicoAsunto(s)
Epilepsia Refractaria , Epilepsia , Estado Epiléptico , Humanos , Florida , Estado Epiléptico/terapiaRESUMEN
Inflammatory response of blood-brain barrier (BBB) endothelial cells plays an important role in pathogenesis of many central nervous system inflammatory diseases, including multiple sclerosis; however, the molecular mechanism mediating BBB endothelial cell inflammatory response remains unclear. In this study, we first observed that knockdown of neuropilin-1 (NRP1), a co-receptor of several structurally diverse ligands, suppressed interferon-γ (IFNγ)-induced C-X-C motif chemokine 10 expression and activation of STAT1 in brain microvascular endothelial cells in a Rac1-dependent manner. Moreover, endothelial-specific NRP1-knockout mice, VECadherin-Cre-ERT2/NRP1flox/flox mice, showed attenuated disease progression during experimental autoimmune encephalomyelitis, a mouse neuroinflammatory disease model. Detailed analysis utilizing histological staining, quantitative PCR, flow cytometry and magnetic resonance imaging demonstrated that deletion of endothelial NRP1 suppressed neuron demyelination, altered lymphocyte infiltration, preserved BBB function and decreased activation of the STAT1-CXCL10 pathway. Furthermore, increased expression of NRP1 was observed in endothelial cells of acute multiple sclerosis lesions. Our data identify a new molecular mechanism of brain microvascular endothelial inflammatory response through NRP1-IFNγ crosstalk that could be a potential target for intervention of endothelial cell dysfunction in neuroinflammatory diseases.
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Encéfalo/irrigación sanguínea , Células Endoteliales/metabolismo , Interferón gamma/farmacología , Microvasos/citología , Neuropilina-1/metabolismo , Transducción de Señal/efectos de los fármacos , Animales , Barrera Hematoencefálica/patología , Quimiocina CXCL10 , Modelos Animales de Enfermedad , Progresión de la Enfermedad , Encefalomielitis Autoinmune Experimental/patología , Células Endoteliales/efectos de los fármacos , Eliminación de Gen , Técnicas de Silenciamiento del Gen , Humanos , Inflamación/patología , Ratones Endogámicos C57BL , Esclerosis Múltiple/metabolismo , Esclerosis Múltiple/patología , Factor de Transcripción STAT1/metabolismo , Regulación hacia Arriba/efectos de los fármacos , Proteína de Unión al GTP rac1/metabolismoRESUMEN
OBJECTIVES: Although secondary hemophagocytic lymphohistiocytosis (HLH) has been reported in children with critical illness of various etiologies, it has not been reported in patients with febrile infection-related epilepsy syndrome (FIRES). We describe a series of patients with concurrent HLH and FIRES in an effort to establish common pathophysiologic abnormalities. METHODS: Five patients with FIRES who were assessed for HLH were identified from a neurocritical care database. All were previously healthy and had extensive diagnostic testing. All had clinical deterioration with multiorgan dysfunction prompting HLH screening 20-29 days after hospitalization. Markers for inflammatory dysregulation were assessed in cerebrospinal fluid (CSF) and serum at various time points. Outcomes were assessed 6 months after presentation. RESULTS: Three patients met clinical criteria for secondary HLH. Elevation of specific cytokines/chemokines was variable. CSF neopterin, high mobility group box 1 (HMGB1), and C-X-C motif chemokine ligand 8 (CXCL8) were significantly elevated in all. Interleukin-1ß (IL-1ß) and IL-18 were not elevated in any of the samples. Treatment and outcomes were variable. SIGNIFICANCE: We describe 3 patients with HLH and FIRES. The co-occurrence of these 2 rare disorders suggests the possibility of a common immune dysregulation phenotype prolonging epileptogenesis. HLH screening in critically ill patients with FIRES may yield a broader understanding of shared inflammatory processes.
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Linfohistiocitosis Hemofagocítica/complicaciones , Linfohistiocitosis Hemofagocítica/diagnóstico , Convulsiones Febriles/complicaciones , Antiinflamatorios/uso terapéutico , Niño , Preescolar , Trastornos del Conocimiento/etiología , Enfermedad Crítica , Citocinas/sangre , Citocinas/líquido cefalorraquídeo , Citocinas/metabolismo , Femenino , Estudios de Seguimiento , Proteína HMGB1/líquido cefalorraquídeo , Humanos , Factores Inmunológicos/uso terapéutico , Linfohistiocitosis Hemofagocítica/terapia , Masculino , Metilprednisolona/uso terapéutico , Neopterin/líquido cefalorraquídeo , Convulsiones Febriles/terapiaRESUMEN
BACKGROUND: Viral encephalitis is a dangerous compromise between the need to robustly clear pathogen from the brain and the need to protect neurons from bystander injury. Theiler's murine encephalomyelitis virus (TMEV) infection of C57Bl/6 mice is a model of viral encephalitis in which the compromise results in hippocampal damage and permanent neurological sequelae. We previously identified brain-infiltrating inflammatory monocytes as the primary driver of this hippocampal pathology, but the mechanisms involved in recruiting these cells to the brain were unclear. METHODS: Chemokine expression levels in the hippocampus were assessed by microarray, ELISA, RT-PCR, and immunofluorescence. Monocyte infiltration during acute TMEV infection was measured by flow cytometry. CCL2 levels were manipulated by immunodepletion and by specific removal from neurons in mice generated by crossing a line expressing the Cre recombinase behind the synapsin promoter to animals with floxed CCL2. RESULTS: Inoculation of the brain with TMEV induced hippocampal production of the proinflammatory chemokine CCL2 that peaked at 6 h postinfection, whereas inoculation with UV-inactivated TMEV did not elicit this response. Immunofluorescence revealed that hippocampal neurons expressed high levels of CCL2 at this timepoint. Genetic deletion of CCR2 and systemic immunodepletion of CCL2 abrogated or blunted the infiltration of inflammatory monocytes into the brain during acute infection. Specific genetic deletion of CCL2 from neurons reduced serum and hippocampal CCL2 levels and inhibited inflammatory monocyte infiltration into the brain. CONCLUSIONS: We conclude that intracranial inoculation with infectious TMEV rapidly induces the expression of CCL2 in neurons, and this cellular source is necessary for CCR2-dependent infiltration of inflammatory monocytes into the brain during the most acute stage of encephalitis. These findings highlight a unique role for neuronal production of chemokines in the initiation of leukocytic infiltration into the infected central nervous system.
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Quimiocina CCL2/biosíntesis , Encefalitis Viral/mortalidad , Hipocampo/patología , Monocitos/inmunología , Neuronas/metabolismo , Animales , Infecciones por Cardiovirus/inmunología , Infecciones por Cardiovirus/metabolismo , Infecciones por Cardiovirus/patología , Quimiotaxis de Leucocito/inmunología , Encefalitis Viral/inmunología , Encefalitis Viral/metabolismo , Encefalitis Viral/patología , Hipocampo/inmunología , Hipocampo/virología , Ratones , Ratones Endogámicos C57BL , TheilovirusRESUMEN
Febrile infection-related epilepsy syndrome (FIRES) is a devastating epileptic encephalopathy with limited treatment options and an unclear etiology. Anakinra is a recombinant version of the human interleukin-1 receptor antagonist used to treat autoinflammatory disorders. This is the first report of anakinra for treatment of a child with super-refractory status epilepticus secondary to FIRES. Anakinra was well tolerated and effective. Cerebral spinal fluid analysis revealed elevated levels of proinflammatory cytokines before treatment that normalized on anakinra, suggesting a potential pathogenic role for neuroinflammation in FIRES. Further studies are required to assess anakinra efficacy and dosing, and to further delineate disease etiology. Ann Neurol 2016;80:939-945.
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Encefalitis Infecciosa/complicaciones , Proteína Antagonista del Receptor de Interleucina 1/uso terapéutico , Convulsiones Febriles/complicaciones , Estado Epiléptico/complicaciones , Estado Epiléptico/tratamiento farmacológico , Preescolar , Femenino , Humanos , Encefalitis Infecciosa/líquido cefalorraquídeo , Encefalitis Infecciosa/tratamiento farmacológico , Mediadores de Inflamación/líquido cefalorraquídeo , Proteínas Recombinantes/uso terapéutico , Convulsiones Febriles/líquido cefalorraquídeo , Convulsiones Febriles/tratamiento farmacológico , Estado Epiléptico/líquido cefalorraquídeo , SíndromeRESUMEN
Pathogenic autoantibodies associated with neuromyelitis optica (NMO) induce disease by targeting aquaporin-4 (AQP4) water channels enriched on astrocytic endfeet at blood-brain interfaces. AQP4 is also expressed at cerebrospinal fluid (CSF)-brain interfaces, such as the pial glia limitans and the ependyma and at the choroid plexus blood-CSF barrier. However, little is known regarding pathology at these sites in NMO. Therefore, we evaluated AQP4 expression, microglial reactivity, and complement deposition at pial and ependymal surfaces and in the fourth ventricle choroid plexus in 23 autopsy cases with clinically and/or pathologically confirmed NMO or NMO spectrum disorder. These findings were compared to five cases with multiple sclerosis, five cases of choroid plexus papilloma, and five control cases without central nervous system disease. In the NMO cases, AQP4 immunoreactivity was reduced relative to control levels in the pia (91%; 21/23), ependyma (56%; 9/16), and choroid plexus epithelium (100%; 12/12). AQP4 immunoreactivity was normal in MS cases in these regions. Compared to MS, NMO cases also showed a focal pattern of pial and ependymal complement deposition and more pronounced microglial reactivity. In addition, AQP4 loss, microglial reactivity, and complement deposition colocalized along the pia and ependyma only in NMO cases. Within the choroid plexus, AQP4 loss was coincident with C9neo immunoreactivity on epithelial cell membranes only in NMO cases. These observations demonstrate that NMO immunopathology extends beyond perivascular astrocytic foot processes to include the pia, ependyma, and choroid plexus, suggesting that NMO IgG-induced pathological alterations at CSF-brain and blood-CSF interfaces may contribute to the occurrence of ventriculitis, leptomeningitis, and hydrocephalus observed among NMO patients. Moreover, disruption of the blood-CSF barrier induced by binding of NMO IgG to AQP4 on the basolateral surface of choroid plexus epithelial cells may provide a unique portal for entry of the pathogenic antibody into the central nervous system.
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Plexo Coroideo/patología , Epéndimo/patología , Neuromielitis Óptica/patología , Piamadre/patología , Adolescente , Adulto , Anciano , Anciano de 80 o más Años , Acuaporina 4/metabolismo , Astrocitos/metabolismo , Astrocitos/patología , Líquido Cefalorraquídeo , Plexo Coroideo/metabolismo , Estudios de Cohortes , Epéndimo/metabolismo , Femenino , Expresión Génica , Humanos , Masculino , Microglía/metabolismo , Microglía/patología , Persona de Mediana Edad , Neuromielitis Óptica/metabolismo , Piamadre/metabolismo , Médula Espinal/metabolismo , Médula Espinal/patología , Adulto JovenRESUMEN
BACKGROUND: Astrocytes expressing the aquaporin-4 water channel are a primary target of pathogenic, disease-specific immunoglobulins (IgG) found in patients with neuromyelitis optica (NMO). Immunopathological analyses of active NMO lesions highlight a unique inflammatory phenotype marked by infiltration of granulocytes. Previous studies characterized this granulocytic infiltrate as a response to vasculocentric complement activation and localized tissue destruction. In contrast, we observe that granulocytic infiltration in NMO lesions occurs independently of complement-mediated tissue destruction or active demyelination. These immunopathological findings led to the hypothesis that NMO IgG stimulates astrocyte signaling that is responsible for granulocytic recruitment in NMO. METHODS: Histopathology was performed on archival formalin-fixed paraffin-embedded autopsy-derived CNS tissue from 23 patients clinically and pathologically diagnosed with NMO or NMO spectrum disorder. Primary murine astroglial cultures were stimulated with IgG isolated from NMO patients or control IgG from healthy donors. Transcriptional responses were assessed by microarray, and translational responses were measured by ELISA. Signaling through the NFκB pathway was measured by western blotting and immunostaining. RESULTS: Stimulation of primary murine astroglial cultures with NMO IgG elicited a reactive and inflammatory transcriptional response that involved signaling through the canonical NFκB pathway. This signaling resulted in the release of pro-granulocytic chemokines and was inhibited by the clinically relevant proteasome inhibitors bortezomib and PR-957. CONCLUSIONS: We propose that the astrocytic NFκB-dependent inflammatory response to stimulation by NMO IgG represents one of the earliest events in NMO pathogenesis, providing a target for therapeutic intervention upstream of irreversible cell death and tissue damage.
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Granulocitos/efectos de los fármacos , Inmunoglobulina G/farmacología , FN-kappa B/metabolismo , Neuroglía/efectos de los fármacos , Transducción de Señal/efectos de los fármacos , Animales , Animales Recién Nacidos , Antineoplásicos/farmacología , Bortezomib/farmacología , Células Cultivadas , Citocinas/genética , Citocinas/metabolismo , Perfilación de la Expresión Génica , Regulación de la Expresión Génica/efectos de los fármacos , Proteína Ácida Fibrilar de la Glía/metabolismo , Humanos , Inmunoglobulina G/sangre , Ratones , Ratones Endogámicos BALB C , Neuromielitis Óptica/sangre , Neuromielitis Óptica/inmunología , Infiltración Neutrófila/efectos de los fármacos , Oligopéptidos/farmacología , Inhibidores de Proteasoma/farmacologíaRESUMEN
Neuromyelitis optica (NMO) is a primary astrocyte disease associated with central nervous system inflammation, demyelination, and tissue injury. Brain lesions are frequently observed in regions enriched in expression of the aquaporin-4 (AQP4) water channel, an antigenic target of the NMO IgG serologic marker. Based on observations of disease reversibility and careful characterization of NMO lesion development, we propose that the NMO IgG may induce a dynamic immunological response in astrocytes. Using primary rat astrocyte-enriched cultures and treatment with NMO patient-derived serum or purified IgG, we observed a robust pattern of gene expression changes consistent with the induction of a reactive and inflammatory phenotype in astrocytes. The reactive astrocyte factor lipocalin-2 and a broad spectrum of chemokines, cytokines, and stress response factors were induced by either NMO patient serum or purified IgG. Treatment with IgG from healthy controls had no effect. The effect is disease-specific, as serum from patients with relapsing-remitting multiple sclerosis, Sjögren's, or systemic lupus erythematosus did not induce a response in the cultures. We hypothesize that binding of the NMO IgG to AQP4 induces a cellular response that results in transcriptional and translational events within the astrocyte that are consistent with a reactive and inflammatory phenotype. Strategies aimed at reducing the inflammatory response of astrocytes may short circuit an amplification loop associated with NMO lesion development.
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Astrocitos/inmunología , Inmunidad Celular/inmunología , Inmunoglobulina G/inmunología , Neuromielitis Óptica/sangre , Neuromielitis Óptica/inmunología , Animales , Astrocitos/efectos de los fármacos , Células Cultivadas , Humanos , Inmunidad Celular/efectos de los fármacos , Inmunoglobulina G/farmacología , Ratas , Ratas Endogámicas LewRESUMEN
The unique architecture of the brain and the blood-brain barrier imposes challenges for the measurement of parenchyma-derived biomarkers that prevent sufficient understanding of transient neuropathogenic processes. One solution to this challenge is direct sampling of brain interstitial fluid via implanted microperfusion probes. Seeking to understand spatial limitations to microperfusion in the brain, we employed computational fluid dynamics modeling and empirical recovery of fluorescently labeled dextrans in an animal model. We found that dextrans were successfully recovered via microperfusion over a 6 h sampling period, especially at probes implanted 2 mm from the dextran infusion point relative to probes implanted 5 mm from the injection site. Experimental recovery was consistently around 1% of simulated, suggesting that this parameter can be used to set practical limits on the maximal tissue concentration of proteins measured in microperfusates and on the spatial domain sampled by our multimodal microperfusion probe.
RESUMEN
Axon injury is a central determinant of irreversible neurological deficit and disease progression in patients with multiple sclerosis (MS). CD8(+) lymphocytes (CTLs) within inflammatory demyelinated MS lesions correlate with acute axon injury and neurological deficits. The mechanisms of these correlations are unknown. We interrogated CTL-mediated axon injury using the transgenic OT-I antigen-specific CTL model system in conjunction with a chambered cortical neuron culture platform that permitted the isolated manipulation of axons independent of neuron cell bodies and glia. Interferon gamma upregulated, through a dose dependent mechanism, the axonal expression of functional major histocompatibility complex class I (MHC I) molecules competent to present immunologically-relevant antigens derived from endogenously expressed proteins. Antigen-specific CTLs formed cytotoxic immune synapses with and directly injured axons expressing antigen-loaded MHC I molecules. CTL-mediated axon injury was mechanistically dependent upon axonal MHC I antigen presentation, T cell receptor specificity and axoplasmic granzyme B activity. Despite extensive distal CTL-mediated axon injury, acute neuron cell body apoptosis was not observed. These findings present a novel model of immune-mediated axon injury and offer anti-axonal CTLs and granzyme B as targets for the therapeutic protection of axons and prevention of neurological deficits in MS patients.
Asunto(s)
Axones/metabolismo , Antígenos CD8/metabolismo , Granzimas/metabolismo , Antígenos de Histocompatibilidad Clase I/metabolismo , Activación de Linfocitos/inmunología , Linfocitos T Citotóxicos/metabolismo , Animales , Axones/patología , Axones/ultraestructura , Corteza Cerebral/citología , Embrión de Mamíferos , Regulación de la Expresión Génica/efectos de los fármacos , Regulación de la Expresión Génica/inmunología , Granzimas/genética , Antígenos de Histocompatibilidad Clase I/genética , Interferón gamma/farmacología , Ratones , Ratones Endogámicos C57BL , Ratones Transgénicos , Proteínas del Tejido Nervioso/metabolismo , Neuroglía/citología , Neuroglía/ultraestructura , Neuronas/citología , Neuronas/efectos de los fármacos , Neuronas/metabolismo , Ovalbúmina/genética , Ovalbúmina/metabolismo , Ovalbúmina/farmacología , Fragmentos de Péptidos/farmacología , Linfocitos T Citotóxicos/efectos de los fármacos , Linfocitos T Citotóxicos/inmunología , Linfocitos T Citotóxicos/ultraestructuraRESUMEN
OBJECTIVE: Therapeutic strategies for patients with febrile infection-related epilepsy syndrome (FIRES) are limited, ad hoc, and frequently ineffective. Based on evidence that inflammation drives pathogenesis in FIRES, we used ex vivo stimulation of peripheral blood mononuclear cells (PBMCs) to characterize the monocytic response profile before and after therapy in a child successfully treated with dexamethasone delivered intrathecally six times between hospital Day 23 and 40 at 0.25 mg/kg/dose. METHODS: PBMCs were isolated from serial blood draws acquired during refractory status epilepticus (RSE) and following resolution associated with intrathecal dexamethasone therapy in a previously healthy 9-year-old male that presented with seizures following Streptococcal pharyngitis. Cells were stimulated with bacterial or viral ligands and cytokine release was measured and compared to responses in age-matched healthy control PBMCs. Levels of inflammatory factors in the blood and CSF were also measured and compared to pediatric healthy control ranges. RESULTS: During RSE, serum levels of IL6, CXCL8, HMGB1, S100A8/A9, and CRP were significantly elevated. IL6 was elevated in CSF. Ex vivo stimulation of PBMCs collected during RSE revealed hyperinflammatory release of IL6 and CXCL8 in response to bacterial stimulation. Following intrathecal dexamethasone, RSE resolved, inflammatory levels normalized in serum and CSF, and the PBMC hyperinflammatory response renormalized. SIGNIFICANCE: FIRES may be associated with a hyperinflammatory monocytic response to normally banal bacterial pathogens. This hyperinflammatory response may induce a profound neutrophil burden and the consequent release of factors that further exacerbate inflammation and drive neuroinflammation. Intrathecal dexamethasone may resolve RSE by resetting this inflammatory feedback loop.
Asunto(s)
Epilepsia Refractaria , Encefalitis , Estado Epiléptico , Masculino , Humanos , Niño , Leucocitos Mononucleares , Monocitos , Interleucina-6 , Convulsiones/tratamiento farmacológico , Estado Epiléptico/tratamiento farmacológico , Epilepsia Refractaria/tratamiento farmacológico , Encefalitis/complicaciones , Inflamación/complicaciones , Dexametasona/farmacologíaRESUMEN
CD8+ T cells outnumber CD4+ cells in multiple sclerosis (MS) lesions associated with disease progression, but the pathogenic role and antigenic targets of these clonally expanded effectors are unknown. Based on evidence that demyelination is necessary but not sufficient for disease progression in MS, we previously hypothesized that CNS-infiltrating CD8+ T cells specific for neuronal antigens directly drive the axonal and neuronal injury that leads to cumulative neurologic disability in patients with MS. We now show that demyelination induced expression of MHC class I on neurons and axons and resulted in presentation of a neuron-specific neoantigen (synapsin promoter-driven chicken ovalbumin) to antigen-specific CD8+ T cells (anti-ovalbumin OT-I TCR-transgenic T cells). These neuroantigen-specific effectors surveilled the CNS in the absence of demyelination but were not retained. However, upon induction of demyelination via cuprizone intoxication, neuroantigen-specific CD8+ T cells proliferated, accumulated in the CNS, and damaged neoantigen-expressing neurons and axons. We further report elevated neuronal expression of MHC class I and ß2-microglobulin transcripts and protein in gray matter and white matter tracts in tissue from patients with MS. These findings support a pathogenic role for autoreactive anti-axonal and anti-neuronal CD8+ T cells in MS progression.