RESUMEN
Cerebrovascular injury (CVI) is a common pathology caused by infections, injury, stroke, neurodegeneration and autoimmune disease. Rapid resolution of a CVI requires a coordinated innate immune response. In the present study, we sought mechanistic insights into how central nervous system-infiltrating monocytes program resident microglia to mediate angiogenesis and cerebrovascular repair after an intracerebral hemorrhage. In the penumbrae of human stroke brain lesions, we identified a subpopulation of microglia that express vascular endothelial growth factor A. These cells, termed 'repair-associated microglia' (RAMs), were also observed in a rodent model of CVI and coexpressed interleukin (IL)-6Ra. Cerebrovascular repair did not occur in IL-6 knockouts or in mice lacking microglial IL-6Ra expression and single-cell transcriptomic analyses revealed faulty RAM programming in the absence of IL-6 signaling. Infiltrating CCR2+ monocytes were the primary source of IL-6 after a CVI and were required to endow microglia with proliferative and proangiogenic properties. Faulty RAM programming in the absence of IL-6 or inflammatory monocytes resulted in poor cerebrovascular repair, neuronal destruction and sustained neurological deficits that were all restored via exogenous IL-6 administration. These data provide a molecular and cellular basis for how monocytes instruct microglia to repair damaged brain vasculature and promote functional recovery after injury.
Asunto(s)
Monocitos , Accidente Cerebrovascular , Ratones , Humanos , Animales , Microglía , Interleucina-6/genética , Interleucina-6/metabolismo , Factor A de Crecimiento Endotelial Vascular/metabolismo , Accidente Cerebrovascular/patología , Encéfalo/metabolismo , Ratones Endogámicos C57BLRESUMEN
Tissue macrophages have an embryonic origin and can be replenished in some tissues under steady-state conditions by blood monocytes. However, little is known about the residency and properties of infiltrating monocytes after an inflammatory challenge. The meninges of the central nervous system (CNS) are populated by a dense network of macrophages that act as resident immune sentinels. Here we show that, following lymphocytic choriomeningitis virus infection, resident meningeal macrophages (MMs) acquired viral antigen and interacted directly with infiltrating cytotoxic T lymphocytes, which led to macrophage depletion. Concurrently, the meninges were infiltrated by inflammatory monocytes that engrafted the meningeal niche and remained in situ for months after viral clearance. This engraftment led to interferon-γ-dependent functional changes in the pool of MMs, including loss of bacterial and immunoregulatory sensors. Collectively, these data indicate that peripheral monocytes can engraft the meninges after an inflammatory challenge, imprinting the compartment with long-term defects in immune function.
Asunto(s)
Sistema Nervioso Central/inmunología , Macrófagos/inmunología , Meningitis Viral/inmunología , Monocitos/inmunología , Animales , Inmunidad , Inflamación/inmunología , Interferón gamma/fisiología , Meninges/inmunología , RatonesRESUMEN
Multiple sclerosis (MS) lesions that do not resolve in the months after they form harbour ongoing demyelination and axon degeneration, and are identifiable in vivo by their paramagnetic rims on MRI scans1-3. Here, to define mechanisms underlying this disabling, progressive neurodegenerative state4-6 and foster development of new therapeutic agents, we used MRI-informed single-nucleus RNA sequencing to profile the edge of demyelinated white matter lesions at various stages of inflammation. We uncovered notable glial and immune cell diversity, especially at the chronically inflamed lesion edge. We define 'microglia inflamed in MS' (MIMS) and 'astrocytes inflamed in MS', glial phenotypes that demonstrate neurodegenerative programming. The MIMS transcriptional profile overlaps with that of microglia in other neurodegenerative diseases, suggesting that primary and secondary neurodegeneration share common mechanisms and could benefit from similar therapeutic approaches. We identify complement component 1q (C1q) as a critical mediator of MIMS activation, validated immunohistochemically in MS tissue, genetically by microglia-specific C1q ablation in mice with experimental autoimmune encephalomyelitis, and therapeutically by treating chronic experimental autoimmune encephalomyelitis with C1q blockade. C1q inhibition is a potential therapeutic avenue to address chronic white matter inflammation, which could be monitored by longitudinal assessment of its dynamic biomarker, paramagnetic rim lesions, using advanced MRI methods.
Asunto(s)
Astrocitos/patología , Linfocitos/patología , Microglía/patología , Esclerosis Múltiple/patología , Animales , Encéfalo/patología , Complemento C1q/antagonistas & inhibidores , Complemento C1q/metabolismo , Encefalomielitis Autoinmune Experimental/patología , Femenino , Humanos , Inflamación/patología , Imagen por Resonancia Magnética , Masculino , Ratones , Ratones Endogámicos C57BL , Persona de Mediana Edad , Esclerosis Múltiple/diagnóstico por imagen , RNA-Seq , Transcriptoma , Sustancia Blanca/patologíaRESUMEN
Regional cellular heterogeneity is a fundamental feature of the human neocortex; however, details of this heterogeneity are still undefined. We used single-nucleus RNA-sequencing to examine cell-specific transcriptional features in the dorsolateral PFC (DLPFC) and the subgenual anterior cingulate cortex (sgACC), regions implicated in major psychiatric disorders. Droplet-based nuclei-capture and library preparation were performed on replicate samples from 8 male donors without history of psychiatric or neurologic disorder. Unsupervised clustering identified major neural cell classes. Subsequent iterative clustering of neurons further revealed 20 excitatory and 22 inhibitory subclasses. Inhibitory cells were consistently more abundant in the sgACC and excitatory neuron subclusters exhibited considerable variability across brain regions. Excitatory cell subclasses also exhibited greater within-class transcriptional differences between the two regions. We used these molecular definitions to determine which cell classes might be enriched in loci carrying a genetic signal in genome-wide association studies or for differentially expressed genes in mental illness. We found that the heritable signals of psychiatric disorders were enriched in neurons and that, while the gene expression changes detected in bulk-RNA-sequencing studies were dominated by glial cells, some alterations could be identified in specific classes of excitatory and inhibitory neurons. Intriguingly, only two excitatory cell classes exhibited concomitant region-specific enrichment for both genome-wide association study loci and transcriptional dysregulation. In sum, by detailing the molecular and cellular diversity of the DLPFC and sgACC, we were able to generate hypotheses on regional and cell-specific dysfunctions that may contribute to the development of mental illness.SIGNIFICANCE STATEMENT Dysfunction of the subgenual anterior cingulate cortex has been implicated in mood disorders, particularly major depressive disorder, and the dorsolateral PFC, a subsection of the PFC involved in executive functioning, has been implicated in schizophrenia. Understanding the cellular composition of these regions is critical to elucidating the neurobiology underlying psychiatric and neurologic disorders. We studied cell type diversity of the subgenual anterior cingulate cortex and dorsolateral PFC of humans with no neuropsychiatric illness using a clustering analysis of single-nuclei RNA-sequencing data. Defining the transcriptomic profile of cellular subpopulations in these cortical regions is a first step to demystifying the cellular and molecular pathways involved in psychiatric disorders.
Asunto(s)
Trastorno Depresivo Mayor , Corteza Prefontal Dorsolateral , Humanos , Masculino , Trastorno Depresivo Mayor/metabolismo , Giro del Cíngulo/metabolismo , Corteza Prefrontal/fisiología , Estudio de Asociación del Genoma Completo , Núcleo Solitario/metabolismoRESUMEN
INTRODUCTION: Variants of uncertain significance (VUS) surged with affordable genetic testing, posing challenges for determining pathogenicity. We examine the pathogenicity of a novel VUS P93S in Annexin A11 (ANXA11) - an amyotrophic lateral sclerosis/frontotemporal dementia-associated gene - in a corticobasal syndrome kindred. Established ANXA11 mutations cause ANXA11 aggregation, altered lysosomal-RNA granule co-trafficking, and transactive response DNA binding protein of 43 kDa (TDP-43) mis-localization. METHODS: We described the clinical presentation and explored the phenotypic diversity of ANXA11 variants. P93S's effect on ANXA11 function and TDP-43 biology was characterized in induced pluripotent stem cell-derived neurons alongside multiomic neuronal and microglial profiling. RESULTS: ANXA11 mutations were linked to corticobasal syndrome cases. P93S led to decreased lysosome colocalization, neuritic RNA, and nuclear TDP-43 with cryptic exon expression. Multiomic microglial signatures implicated immune dysregulation and interferon signaling pathways. DISCUSSION: This study establishes ANXA11 P93S pathogenicity, broadens the phenotypic spectrum of ANXA11 mutations, underscores neuronal and microglial dysfunction in ANXA11 pathophysiology, and demonstrates the potential of cellular models to determine variant pathogenicity. HIGHLIGHTS: ANXA11 P93S is a pathogenic variant. Corticobasal syndrome is part of the ANXA11 phenotypic spectrum. Hybridization chain reaction fluorescence in situ hybridization (HCR FISH) is a new tool for the detection of cryptic exons due to TDP-43-related loss of splicing regulation. Microglial ANXA11 and related immune pathways are important drivers of disease. Cellular models are powerful tools for adjudicating variants of uncertain significance.
Asunto(s)
Anexinas , Proteínas de Unión al ADN , Humanos , Proteínas de Unión al ADN/genética , Proteínas de Unión al ADN/metabolismo , Anexinas/genética , Masculino , Mutación/genética , Femenino , Esclerosis Amiotrófica Lateral/genética , Esclerosis Amiotrófica Lateral/patología , Neuronas/metabolismo , Neuronas/patología , Demencia Frontotemporal/genética , Demencia Frontotemporal/patología , Persona de Mediana Edad , AncianoRESUMEN
Ionizing radiation-induced tissue damage recruits monocytes into the exposed area where they are differentiated to macrophages. These implement phagocytic removal of dying cells and elicit an acute inflammatory response, but can also facilitate tumorigenesis due to production of anti-inflammatory cytokines. Using primary human monocyte-derived macrophages (MDMs) and the THP1 monocytic cell line, we demonstrate that gamma radiation triggers monocyte differentiation toward the macrophage phenotype with increased expression of type I interferons (IFN-I) and both pro- and anti-inflammatory macrophage activation markers. We found that these changes correlate with significantly upregulated expression of 622 retroelements from various groups, particularly of several clades of human endogenous retroviruses (HERVs). Elevated transcription was detected in both sense and antisense directions in the HERV subgroups tested, including the most genetically homogeneous clade HML-2. The level of antisense transcription was three- to five-fold higher than of the sense strand levels. Using a proximity ligation assay and immunoprecipitation followed by RNA quantification, we identified an increased amount of the dsRNA receptors MDA-5 and TLR3 bound to an equivalent number of copies of sense and antisense chains of HERVK HML-2 RNA. This binding triggered MAVS-associated signaling pathways resulting in increased expression of IFN-I and inflammation related genes that enhanced the cumulative inflammatory effect of radiation-induced senescence. HML-2 knockdown was accompanied with reduced expression and secretion of IFNα, pro-inflammatory (IL-1ß, IL-6, CCL2, CCL3, CCL8, and CCL20) and anti-inflammatory (IL10) modulators in irradiated monocytes and MDMs. Taken together, our data indicate that radiation stress-induced HERV expression enhances the IFN-I and cytokine response and results in increased levels of pro-inflammatory modulators along with expression of anti-inflammatory factors associated with the macrophage tumorigenic phenotype.
Asunto(s)
Retrovirus Endógenos/genética , Rayos gamma , Inflamación/inmunología , Activación de Macrófagos/inmunología , Macrófagos/inmunología , Monocitos/inmunología , Retroelementos/genética , Diferenciación Celular , Citocinas/metabolismo , Humanos , Inflamación/metabolismo , Inflamación/patología , Macrófagos/metabolismo , Macrófagos/efectos de la radiación , Monocitos/metabolismo , Monocitos/efectos de la radiación , TranscriptomaRESUMEN
Stem cells are capable of unlimited proliferation but can be induced to form brain cells. Factors that specifically regulate human development are poorly understood. We found that human stem cells expressed high levels of the envelope protein of an endogenized human-specific retrovirus (HERV-K, HML-2) from loci in chromosomes 12 and 19. The envelope protein was expressed on the cell membrane of the stem cells and was critical in maintaining the stemness via interactions with CD98HC, leading to triggering of human-specific signaling pathways involving mammalian target of rapamycin (mTOR) and lysophosphatidylcholine acyltransferase (LPCAT1)-mediated epigenetic changes. Down-regulation or epigenetic silencing of HML-2 env resulted in dissociation of the stem cell colonies and enhanced differentiation along neuronal pathways. Thus HML-2 regulation is critical for human embryonic and neurodevelopment, while it's dysregulation may play a role in tumorigenesis and neurodegeneration.
Asunto(s)
Diferenciación Celular , Retrovirus Endógenos/fisiología , Neuronas/metabolismo , Transducción de Señal , Células Madre/metabolismo , Serina-Treonina Quinasas TOR/metabolismo , Biomarcadores , Diferenciación Celular/genética , Autorrenovación de las Células/genética , Cadena Pesada de la Proteína-1 Reguladora de Fusión/metabolismo , Regulación Viral de la Expresión Génica , Humanos , Células Madre Pluripotentes Inducidas/citología , Células Madre Pluripotentes Inducidas/metabolismo , Células-Madre Neurales/citología , Células-Madre Neurales/metabolismo , Neuronas/citología , Unión Proteica , Células Madre/citología , Factores de Transcripción/genética , Factores de Transcripción/metabolismo , Proteínas del Envoltorio Viral/genéticaRESUMEN
Small ubiquitin-like modifier (SUMO1-3) conjugation (SUMOylation), a posttranslational modification, modulates almost all major cellular processes. Mounting evidence indicates that SUMOylation plays a crucial role in maintaining and regulating neural function, and importantly its dysfunction is implicated in cognitive impairment in humans. We have previously shown that simultaneously silencing SUMO1-3 expression in neurons negatively affects cognitive function. However, the roles of the individual SUMOs in modulating cognition and the mechanisms that link SUMOylation to cognitive processes remain unknown. To address these questions, in this study, we have focused on SUMO2 and generated a new conditional Sumo2 knockout mouse line. We found that conditional deletion of Sumo2 predominantly in forebrain neurons resulted in marked impairments in various cognitive tests, including episodic and fear memory. Our data further suggest that these abnormalities are attributable neither to constitutive changes in gene expression nor to alterations in neuronal morphology, but they involve impairment in dynamic SUMOylation processes associated with synaptic plasticity. Finally, we provide evidence that dysfunction on hippocampal-based cognitive tasks was associated with a significant deficit in the maintenance of hippocampal long-term potentiation in Sumo2 knockout mice. Collectively, these data demonstrate that protein conjugation by SUMO2 is critically involved in cognitive processes.
Asunto(s)
Memoria , Proteínas Modificadoras Pequeñas Relacionadas con Ubiquitina/metabolismo , Animales , Cognición , Femenino , Hipocampo/metabolismo , Hipocampo/fisiología , Potenciación a Largo Plazo , Masculino , Ratones , Ratones Endogámicos C57BL , Prosencéfalo/metabolismo , Prosencéfalo/fisiología , Proteínas Modificadoras Pequeñas Relacionadas con Ubiquitina/genéticaRESUMEN
BACKGROUND: Thymic hypoplasia/aplasia occurs as a part of DiGeorge syndrome, which has several known genetic causes, and with loss-of-function mutations in forkhead box N1 (FOXN1). OBJECTIVE: We sought to determine the cause of selective T-cell lymphopenia with inverted kappa/lambda ratio in several kindreds. METHODS: Patients were identified through newborn screening for severe combined immunodeficiency using the T-cell receptor excision circle assay. Those found to have selective T-cell lymphopenia underwent testing with chromosomal microarray analysis. Three-week-old mice heterozygous for a loss-of-function mutation in forkhead box I3 (FOXI3), a candidate gene within the common deleted region found in patients, were compared with wild-type littermates. Assessments included body and organ weights, flow cytometric analysis of thymocytes and splenocytes, and histologic/transcriptomic analyses of thymic tissue. RESULTS: Five kindreds with similar immunophenotypes that included selective T-cell lymphopenia had overlapping microdeletions at chromosome 2p11.2 that spanned FOXI3 and, in most cases, the immunoglobulin kappa light chain locus. Studies in a mouse knockout strain for FOXI3 revealed smaller body weights and relatively lower thymus weights in heterozygous compared with wild-type animals. Histology and flow cytometry on spleens and thymi from 3-week-old pups for T- and B-cell subsets and epithelial cells did not show any significant qualitative or quantitative differences. Transcriptomic analysis of thymic RNA revealed divergence in global transcriptomic signatures, and Ingenuity Pathway Analysis revealed predicted dysfunction in epithelial adherens junctions. CONCLUSIONS: Microdeletions at chromosome 2p11.2 are associated with T-cell lymphopenia and probable thymic hypoplasia in human subjects, and haploinsufficiency for FOXI3, a candidate gene within the deleted region, is the likely underlying cause.
Asunto(s)
Deleción Cromosómica , Cromosomas Humanos Par 2/genética , Síndrome de DiGeorge/genética , Factores de Transcripción Forkhead/genética , Mutación con Pérdida de Función , Animales , Cromosomas Humanos Par 2/inmunología , Síndrome de DiGeorge/inmunología , Síndrome de DiGeorge/patología , Femenino , Factores de Transcripción Forkhead/inmunología , Humanos , Masculino , Ratones , Ratones Mutantes , Timo/inmunología , Timo/patologíaRESUMEN
Methylprednisolone (MP) is an anti-inflammatory drug approved for the treatment of acute spinal cord injuries (SCIs). However, MP administration for SCIs has become a controversial issue while the molecular effects of MP remain unexplored to date. Therefore, delineating the benefits and side effects of MP and determining what MP cannot cure in SCIs at the molecular level are urgent issues. Here, genomic profiles of the spinal cord in rats with and without injury insults, and those with and without MP treatment, were generated at 0, 2, 4, 6, 8, 12, 24, and 48 h post-injury. A comprehensive analysis was applied to obtain three distinct classes: side effect of MP (SEMP), competence of MP (CPMP), and incapability of MP (ICMP). Functional analysis using these genes suggested that MP exerts its greatest effect at 8~12 h, and the CPMP was reflected in the immune response, while SEMP suggested aspects of metabolism, such as glycolysis, and ICMP was on neurological system processes in acute SCIs. For the first time, we are able to precisely reveal responsive functions of MP in SCIs at the molecular level and provide useful solutions to avoid complications of MP in SCIs before better therapeutic drugs are available.
Asunto(s)
Antiinflamatorios/farmacología , Metilprednisolona/farmacología , Traumatismos de la Médula Espinal/patología , Transcriptoma/efectos de los fármacos , Animales , Antiinflamatorios/uso terapéutico , Modelos Animales de Enfermedad , Femenino , Metilprednisolona/uso terapéutico , Ratas , Ratas Long-Evans , Médula Espinal/metabolismo , Traumatismos de la Médula Espinal/tratamiento farmacológico , Factores de TiempoRESUMEN
OBJECTIVE: To determine the underlying etiology in a patient with progressive dementia with extrapyramidal signs and chronic inflammation referred to the National Institutes of Health Undiagnosed Diseases Program. METHODS: Extensive investigations included metabolic profile, autoantibody panel, infectious etiologies, genetic screening, whole exome sequencing, and the phage-display assay, VirScan, for viral immune responses. An etiological diagnosis was established postmortem. RESULTS: Using VirScan, enrichment of dengue viral antibodies was detected in cerebrospinal fluid as compared to serum. No virus was detected in serum or cerebrospinal fluid, but postmortem analysis confirmed dengue virus in the brain by immunohistochemistry, in situ hybridization, quantitative polymerase chain reaction, and sequencing. Dengue virus was also detectable by polymerase chain reaction and sequencing from brain biopsy tissue collected 33 months antemortem, confirming a chronic infection despite a robust immune response directed against the virus. Immunoprofiling and whole exome sequencing of the patient did not reveal any immunodeficiency, and sequencing of the virus demonstrated wild-type dengue virus in the central nervous system. INTERPRETATION: Dengue virus is the most common arbovirus worldwide and represents a significant public health concern. Infections with dengue virus are usually self-limiting, and chronic dengue infections have not been previously reported. Our findings suggest that dengue virus infections may persist in the central nervous system causing a panencephalitis and should be considered in patients with progressive dementia with extrapyramidal features in endemic regions or with relevant travel history. Furthermore, this work highlights the utility of comprehensive antibody profiling assays to aid in the diagnosis of encephalitis of unknown etiology. ANN NEUROL 2019;86:695-703.
Asunto(s)
Dengue/complicaciones , Dengue/patología , Encefalitis Viral/etiología , Encefalitis Viral/patología , Enfermedad Crónica , Demencia , Virus del Dengue , Resultado Fatal , Humanos , Masculino , Persona de Mediana EdadRESUMEN
Alternative splicing (AS) of pre-messenger (m)RNA is a pivotal mechanism in expanding proteomic diversity, which determines the functions of mammalian cells. By conducting transcriptome analyses to profile splicing events in human colorectal cancer (CRC) tissues compared to adjacent normal counterparts, we noted differential splicing profiles of serine/arginine-rich splicing factor 3 (SRSF3) and mitogen-activated protein 4 kinase 4 (MAP4K4) in cancerous tissues of CRC compared to adjacent normal tissues. In addition to SRSF3-mediated autoregulation, RNA-binding motif protein 4 (RBM4) constituted another mechanism in reprogramming the splicing profile of SRSF3. Upregulated expressions of SRSF3 in CRC cells modulated utilization of MAP4K4 exon 16 in a sequence-dependent manner. Alternatively spliced MAP4K4 variants exhibited differential effects on the phosphorylation of c-Jun N-terminal protein kinase 1 (JNK1) which subsequently modulated expression profiles of E-cadherin, N-cadherin, and vimentin, all of which are involved in the migration and invasion of CRC cells. Collectively, RBM4-SRSF3-MAP4K4 constitutes a novel mechanism for manipulating the metastasis of CRC cells through the JNK1 signaling pathway.
Asunto(s)
Neoplasias Colorrectales/metabolismo , Péptidos y Proteínas de Señalización Intracelular/metabolismo , Sistema de Señalización de MAP Quinasas , Proteínas de Neoplasias/metabolismo , Proteínas Serina-Treonina Quinasas/metabolismo , Empalme del ARN , Proteínas de Unión al ARN/metabolismo , Factores de Empalme Serina-Arginina/metabolismo , Neoplasias Colorrectales/genética , Femenino , Humanos , Péptidos y Proteínas de Señalización Intracelular/genética , Masculino , Metástasis de la Neoplasia , Proteínas de Neoplasias/genética , Proteínas Serina-Treonina Quinasas/genética , Proteínas de Unión al ARN/genética , Factores de Empalme Serina-Arginina/genéticaRESUMEN
Lineage commitment and differentiation of skeletal stem cells/bone marrow stromal cells (SSCs/BMSCs, often called bone marrow-derived "mesenchymal stem/stromal" cells) offer an important opportunity to study skeletal and hematopoietic diseases, and for tissue engineering and regenerative medicine. Currently, many studies in this field have relied on cell lineage tracing methods in mouse models, which have provided a significant advancement in our knowledge of skeletal and hematopoietic stem-cell niches in bone marrow (BM). However, there is a lack of agreement in numerous fundamental areas, including origins of various BM stem-cell niches, cell identities, and their physiological roles in the BM. In order to resolve these issues, we propose a new hypothesis of "paralogous" stem-cell niches (PSNs); that is, progressively altered parallel niches within an individual species throughout the life span of the organism. A putative PSN code seems to be plausible based on analysis of transcriptional signatures in two representative genes that encode Nes-GFP and leptin receptors, which are frequently used to monitor SSC lineage development in BM. Furthermore, we suggest a dynamic paralogous BM niche (PBMN) model that elucidates the coupling and uncoupling mechanisms between BM stem-cell niches and their zones of active regeneration during different developmental stages. Elucidation of these PBMNs would enable us to resolve the existing controversies, thus paving the way to achieving precision regenerative medicine and pharmaceutical applications based on these BM cell resources. Stem Cells 2018;36:11-21.
Asunto(s)
Médula Ósea/metabolismo , Células Madre Hematopoyéticas/metabolismo , Nicho de Células Madre/genética , Células Madre/metabolismo , Diferenciación Celular , Linaje de la Célula , HumanosRESUMEN
The intestinal epithelium constitutes a crucial defense to the potentially life-threatening effects of gut microbiota. However, due to a complex underlying vasculature, hypoperfusion and resultant tissue ischemia pose a particular risk to function and integrity of the epithelium. The small ubiquitin-like modifier (SUMO) conjugation pathway critically regulates adaptive responses to metabolic stress and is of particular significance in the gut, as inducible knockout of the SUMO-conjugating enzyme Ubc9 results in rapid intestinal epithelial disintegration. Here we analyzed the pattern of individual SUMO isoforms in intestinal epithelium and investigated their roles in intestinal ischemia/reperfusion (I/R) damage. Immunostaining revealed that epithelial SUMO2/3 expression was almost exclusively limited to crypt epithelial nuclei in unchallenged mice. However, intestinal I/R or overexpression of Ubc9 caused a remarkable enhancement of epithelial SUMO2/3 staining along the crypt-villus axis. Unexpectedly, a similar pattern was found in SUMO1 knockout mice. Ubc9 transgenic mice, but also SUMO1 knockout mice were protected from I/R injury as evidenced by better preserved barrier function and blunted inflammatory responses. PCR array analysis of microdissected villus-tip epithelia revealed a specific epithelial contribution to reduced inflammatory responses in Ubc9 transgenic mice, as key chemotactic signaling molecules such as IL17A were significantly downregulated. Together, our data indicate a critical role particularly of the SUMO2/3 isoforms in modulating responses to I/R and provide the first evidence that SUMO1 deletion activates a compensatory process that protects from ischemic damage.
Asunto(s)
Mucosa Intestinal/irrigación sanguínea , Daño por Reperfusión/prevención & control , Proteína SUMO-1/fisiología , Enzimas Ubiquitina-Conjugadoras/fisiología , Animales , Quimiocinas/análisis , Mucosa Intestinal/química , Captura por Microdisección con Láser , Ratones , Ratones Endogámicos C57BL , Ratones Noqueados , Proteína SUMO-1/deficiencia , Proteínas Modificadoras Pequeñas Relacionadas con Ubiquitina/análisis , Proteínas Modificadoras Pequeñas Relacionadas con Ubiquitina/fisiología , Enzimas Ubiquitina-Conjugadoras/genética , Ubiquitinas/análisis , Ubiquitinas/fisiologíaRESUMEN
BACKGROUND: Inherited Retinal dystrophy (IRD) is a broad group of inherited retinal disorders with heterogeneous genotypes and phenotypes. Next generation sequencing (NGS) methods have been broadly applied for analyzing patients with IRD. Here we report a novel approach to enrich the target gene panel by microdroplet PCR. METHODS: This assay involved a primer library which targeted 3071 amplicons from 2078 exons comprised of 184 genes involved in retinal function and/or retinal development. We amplified the target regions using the RainDance target enrichment PCR method and sequenced the products using the MiSeq NGS platform. RESULTS: In this study, we analyzed 82 samples from 67 families with IRD. Bioinformatics analysis indicated that this procedure was able to reach 99% coverage of target sequences with an average sequence depth of reads at 119×. The variants detected by this study were filtered, validated, and prioritized by pathogenicity analysis. Genotypes and phenotypes were correlated by determining a consistent relationship in 38 propands (56.7%). Pathogenic variants in genes related to retinal function were found in another 11 probands (16.4%), but the clinical correlations showed inconsistencies and insufficiencies in these patients. CONCLUSIONS: The application of NGS in IRD clinical molecular diagnosis provides a powerful approach to exploring the etiology and pathology in patients. It is important for the clinical laboratory to interpret the molecular findings in the context of patient clinical presentations because accurate interpretation of pathogenic variants is critical for delivering solid clinical molecular diagnosis to clinicians and patients and improving the standard care of patients.
Asunto(s)
Análisis Mutacional de ADN/métodos , Mutación , Reacción en Cadena de la Polimerasa/métodos , Distrofias Retinianas/genética , Adulto , Anciano , Femenino , Predisposición Genética a la Enfermedad , Humanos , Masculino , Persona de Mediana Edad , Distrofias Retinianas/diagnósticoRESUMEN
The efficiency of central nervous system remyelination declines with age. This is in part due to an age-associated decline in the phagocytic removal of myelin debris, which contains inhibitors of oligodendrocyte progenitor cell differentiation. In this study, we show that expression of genes involved in the retinoid X receptor pathway are decreased with ageing in both myelin-phagocytosing human monocytes and mouse macrophages using a combination of in vivo and in vitro approaches. Disruption of retinoid X receptor function in young macrophages, using the antagonist HX531, mimics ageing by reducing myelin debris uptake. Macrophage-specific RXRα (Rxra) knockout mice revealed that loss of function in young mice caused delayed myelin debris uptake and slowed remyelination after experimentally-induced demyelination. Alternatively, retinoid X receptor agonists partially restored myelin debris phagocytosis in aged macrophages. The agonist bexarotene, when used in concentrations achievable in human subjects, caused a reversion of the gene expression profile in multiple sclerosis patient monocytes to a more youthful profile and enhanced myelin debris phagocytosis by patient cells. These results reveal the retinoid X receptor pathway as a positive regulator of myelin debris clearance and a key player in the age-related decline in remyelination that may be targeted by available or newly-developed therapeutics.
Asunto(s)
Envejecimiento/metabolismo , Envejecimiento/patología , Vaina de Mielina/metabolismo , Fagocitosis , Receptor alfa X Retinoide/metabolismo , Adulto , Animales , Benzoatos/farmacología , Bexaroteno , Compuestos de Bifenilo/farmacología , Femenino , Humanos , Macrófagos/citología , Macrófagos/efectos de los fármacos , Macrófagos/metabolismo , Masculino , Ratones , Ratones Noqueados , Persona de Mediana Edad , Monocitos/citología , Monocitos/efectos de los fármacos , Monocitos/metabolismo , Esclerosis Múltiple/metabolismo , Fagocitosis/efectos de los fármacos , Receptor alfa X Retinoide/agonistas , Receptor alfa X Retinoide/antagonistas & inhibidores , Receptor alfa X Retinoide/genética , Transducción de Señal/fisiología , Tetrahidronaftalenos/farmacología , Transcriptoma/efectos de los fármacos , Adulto JovenRESUMEN
Chronic immune activation is a major complication of antiretroviral therapy (ART) for HIV infection and can cause a devastating immune reconstitution inflammatory syndrome (IRIS) in the brain. The mechanism of T-cell activation in this population is not well understood. We found HIV-Tat protein and IL-17-expressing mononuclear cells in the brain of an individual with IRIS. Tat was also present in the CSF of individuals virologically controlled on ART. Hence we examined if Tat protein could directly activate T cells. Tat transcriptionally dysregulated 94 genes and induced secretion of 11 cytokines particularly activation of IL-17 signaling pathways supporting the development of a proinflammatory state. Tat increased IL-17 transcription and secretion in T cells. Tat entered the T cells rapidly by clathrin-mediated endocytosis and localized to both the cytoplasm and the nucleus. Tat activated T cells through a nonclassical pathway dependent upon vascular endothelial growth factor receptor-2 and downstream secondary signaling pathways but independent of the T-cell receptor. However, Tat stimulation of T cells did not induce T-cell proliferation but increased viral infectivity. This study demonstrates Tat's role as a virulence factor, by driving T-cell activation and contributing to IRIS pathophysiology. This supports the necessity of an anti-Tat therapy in conjunction with ART and identifies multiple targetable pathways to prevent Tat-mediated T-cell activation.
Asunto(s)
Antirretrovirales/efectos adversos , Regulación Viral de la Expresión Génica/inmunología , Infecciones por VIH/tratamiento farmacológico , Síndrome Inflamatorio de Reconstitución Inmune/fisiopatología , Transducción de Señal/inmunología , Factores de Virulencia/metabolismo , Productos del Gen tat del Virus de la Inmunodeficiencia Humana/metabolismo , Antirretrovirales/uso terapéutico , Encéfalo/metabolismo , Ensayo de Inmunoadsorción Enzimática , Citometría de Flujo , Humanos , Síndrome Inflamatorio de Reconstitución Inmune/etiología , Síndrome Inflamatorio de Reconstitución Inmune/inmunología , Immunoblotting , Interleucina-17/metabolismo , Leucocitos Mononucleares/metabolismo , Activación de Linfocitos/inmunología , Análisis por Micromatrices , Linfocitos T/inmunología , Factores de Virulencia/líquido cefalorraquídeo , Productos del Gen tat del Virus de la Inmunodeficiencia Humana/líquido cefalorraquídeoRESUMEN
Viral infections of central nervous system (CNS) often trigger inflammatory responses that give rise to a wide range of pathological outcomes. The CNS is equipped with an elaborate network of innate immune sentinels (e.g. microglia, macrophages, dendritic cells) that routinely serve as first responders to these infections. The mechanisms that underlie the dynamic programming of these cells following CNS viral infection remain undefined. To gain insights into this programming, we utilized a combination of genomic and two-photon imaging approaches to study a pure innate immune response to a noncytopathic virus (lymphocytic choriomeningitis virus) as it established persistence in the brain. This enabled us to evaluate how global gene expression patterns were translated into myeloid cell dynamics following infection. Two-photon imaging studies revealed that innate myeloid cells mounted a vigorous early response to viral infection characterized by enhanced vascular patrolling and a complete morphological transformation. Interestingly, innate immune activity subsided over time and returned to a quasi-normal state as the virus established widespread persistence in the brain. At the genomic level, early myeloid cell dynamics were associated with massive changes in CNS gene expression, most of which declined over time and were linked to type I interferon signaling (IFN-I). Surprisingly, in the absence of IFN-I signaling, almost no differential gene expression was observed in the nervous system despite increased viral loads. In addition, two-photon imaging studies revealed that IFN-I receptor deficient myeloid cells were unresponsive to viral infection and remained in a naïve state. These data demonstrate that IFN-I engages non-redundant programming responsible for nearly all innate immune activity in the brain following a noncytopathic viral infection. This Achilles' heel could explain why so many neurotropic viruses have acquired strategies to suppress IFN-I.
Asunto(s)
Regulación de la Expresión Génica , Interferón Tipo I/metabolismo , Coriomeningitis Linfocítica/metabolismo , Virus de la Coriomeningitis Linfocítica/metabolismo , Células Mieloides/metabolismo , Proteínas del Tejido Nervioso/biosíntesis , Animales , Interferón Tipo I/genética , Coriomeningitis Linfocítica/genética , Coriomeningitis Linfocítica/patología , Ratones , Ratones Noqueados , Células Mieloides/patología , Proteínas del Tejido Nervioso/genética , Transducción de Señal/genéticaRESUMEN
Salt loading (SL) and water deprivation (WD) are experimental challenges that are often used to study the osmotic circuitry of the brain. Central to this circuit is the supraoptic nucleus (SON) of the hypothalamus, which is responsible for the biosynthesis of the hormones, arginine vasopressin (AVP) and oxytocin (OXT), and their transport to terminals that reside in the posterior lobe of the pituitary. On osmotic challenge evoked by a change in blood volume or osmolality, the SON undergoes a function-related plasticity that creates an environment that allows for an appropriate hormone response. Here, we have described the impact of SL and WD compared with euhydrated (EU) controls in terms of drinking and eating behavior, body weight, and recorded physiological data including circulating hormone data and plasma and urine osmolality. We have also used microarrays to profile the transcriptome of the SON following SL and remined data from the SON that describes the transcriptome response to WD. From a list of 2,783 commonly regulated transcripts, we selected 20 genes for validation by qPCR. All of the 9 genes that have already been described as expressed or regulated in the SON by osmotic stimuli were confirmed in our models. Of the 11 novel genes, 5 were successfully validated while 6 were false discoveries.
Asunto(s)
Cloruro de Sodio Dietético/administración & dosificación , Núcleo Supraóptico/fisiología , Transcriptoma , Privación de Agua , Animales , Arginina Vasopresina/sangre , Volumen Sanguíneo , Peso Corporal , Ingestión de Líquidos , Ingestión de Alimentos , Perfilación de la Expresión Génica/métodos , Regulación de la Expresión Génica , Masculino , Análisis de Secuencia por Matrices de Oligonucleótidos , Concentración Osmolar , Osmorregulación , Oxitocina/sangre , ARN Mensajero/metabolismo , Ratas Sprague-Dawley , Reproducibilidad de los Resultados , Reacción en Cadena de la Polimerasa de Transcriptasa Inversa , Núcleo Supraóptico/metabolismo , Factores de TiempoRESUMEN
A stringent control of homeostasis is critical for functional maintenance and survival of neurons. In the mammalian retina, the basic motif leucine zipper transcription factor NRL determines rod versus cone photoreceptor cell fate and activates the expression of many rod-specific genes. Here, we report an integrated analysis of NRL-centered gene regulatory network by coupling chromatin immunoprecipitation followed by high-throughput sequencing (ChIP-Seq) data from Illumina and ABI platforms with global expression profiling and in vivo knockdown studies. We identified approximately 300 direct NRL target genes. Of these, 22 NRL targets are associated with human retinal dystrophies, whereas 95 mapped to regions of as yet uncloned retinal disease loci. In silico analysis of NRL ChIP-Seq peak sequences revealed an enrichment of distinct sets of transcription factor binding sites. Specifically, we discovered that genes involved in photoreceptor function include binding sites for both NRL and homeodomain protein CRX. Evaluation of 26 ChIP-Seq regions validated their enhancer functions in reporter assays. In vivo knockdown of 16 NRL target genes resulted in death or abnormal morphology of rod photoreceptors, suggesting their importance in maintaining retinal function. We also identified histone demethylase Kdm5b as a novel secondary node in NRL transcriptional hierarchy. Exon array analysis of flow-sorted photoreceptors in which Kdm5b was knocked down by shRNA indicated its role in regulating rod-expressed genes. Our studies identify candidate genes for retinal dystrophies, define cis-regulatory module(s) for photoreceptor-expressed genes and provide a framework for decoding transcriptional regulatory networks that dictate rod homeostasis.