RESUMEN
Myalgic encephalomyelitis/chronic fatigue syndrome (ME/CFS) is a debilitating condition with unknown aetiology, Myalgic encephalomyelitis unclear pathophysiology and with no diagnostic test or biomarker available. Many patients report their ME/CFS began after an acute infection, and subsequent increased frequency of infections, such as colds or influenza, is common. These factors imply an altered immunological status exists in ME/CFS, in at least a proportion of patients, yet previous studies of peripheral immunity have been discrepant and inconclusive. The UK ME/CFS Biobank, which has collected blood samples from nearly 300 clinically-confirmed ME/CFS patients, enables large-scale studies of immunological function in phenotypically well-characterised participants. In this study, herpes virus serological status and T cell, B cell, NK cell and monocyte populations were investigated in 251 ME/CFS patients, including 54 who were severely affected, and compared with those from 107 healthy participants and with 46 patients with Multiple Sclerosis. There were no differences in seroprevalence for six human herpes viruses between ME/CFS and healthy controls, although seroprevalence for the Epstein-Barr virus was higher in multiple sclerosis patients. Contrary to previous reports, no significant differences were observed in NK cell numbers, subtype proportions or in vitro responsiveness between ME/CFS patients and healthy control participants. In contrast, the T cell compartment was altered in ME/CFS, with increased proportions of effector memory CD8+ T cells and decreased proportions of terminally differentiated effector CD8+ T cells. Conversely, there was a significantly increased proportion of mucosal associated invariant T cells (MAIT) cells, especially in severely affected ME/CFS patients. These abnormalities demonstrate that an altered immunological state does exist in ME/CFS, particularly in severely affected people. This may simply reflect ongoing or recent infection, or may indicate future increased susceptibility to infection. Longitudinal studies of ME/CFS patients are needed to help to determine cause and effect and thus any potential benefits of immuno-modulatory treatments for ME/CFS.
Asunto(s)
Síndrome de Fatiga Crónica/inmunología , Inmunidad Celular , Adolescente , Adulto , Anticuerpos Antivirales/sangre , Estudios de Cohortes , Citomegalovirus/inmunología , Femenino , Herpesvirus Humano 4/inmunología , Humanos , Células Asesinas Naturales/inmunología , Leucocitos/inmunología , Masculino , Persona de Mediana Edad , Esclerosis Múltiple , Simplexvirus/inmunología , Linfocitos T/inmunología , Adulto JovenRESUMEN
The immune state of wild animals is largely unknown. Knowing this and what affects it is important in understanding how infection and disease affects wild animals. The immune state of wild animals is also important in understanding the biology of their pathogens, which is directly relevant to explaining pathogen spillover among species, including to humans. The paucity of knowledge about wild animals' immune state is in stark contrast to our exquisitely detailed understanding of the immunobiology of laboratory animals. Making an immune response is costly, and many factors (such as age, sex, infection status, and body condition) have individually been shown to constrain or promote immune responses. But, whether or not these factors affect immune responses and immune state in wild animals, their relative importance, and how they interact (or do not) are unknown. Here, we have investigated the immune ecology of wild house mice-the same species as the laboratory mouse-as an example of a wild mammal, characterising their adaptive humoral, adaptive cellular, and innate immune state. Firstly, we show how immune variation is structured among mouse populations, finding that there can be extensive immune discordance among neighbouring populations. Secondly, we identify the principal factors that underlie the immunological differences among mice, showing that body condition promotes and age constrains individuals' immune state, while factors such as microparasite infection and season are comparatively unimportant. By applying a multifactorial analysis to an immune system-wide analysis, our results bring a new and unified understanding of the immunobiology of a wild mammal.
Asunto(s)
Inmunidad Adaptativa , Infestaciones por Pulgas/inmunología , Inmunidad Humoral , Inmunidad Innata , Infecciones por Nematodos/inmunología , Infestaciones por Garrapatas/inmunología , Animales , Animales Salvajes , Variación Biológica Poblacional/inmunología , Células Dendríticas/citología , Células Dendríticas/inmunología , Ecología , Femenino , Infestaciones por Pulgas/parasitología , Variación Genética/inmunología , Interacciones Huésped-Parásitos/inmunología , Linfocitos/clasificación , Linfocitos/citología , Linfocitos/inmunología , Masculino , Ratones , Análisis Multivariante , Infecciones por Nematodos/parasitología , Estaciones del Año , Infestaciones por Garrapatas/parasitología , Reino UnidoRESUMEN
The UK ME/CFS Biobank was launched in August 2011 following extensive consultation with professionals and patient representatives. The bioresource aims to enhance research on myalgic encephalomyelitis/chronic fatigue syndrome (ME/CFS), related to pathophysiology, biomarkers and therapeutic approaches. The cohort includes 18-60 year olds, encompassing 284 clinically-confirmed ME/CFS cases, 60 neurologist-diagnosed multiple sclerosis (MS) cases, and 135 healthy individuals. The Biobank contains blood samples, aliquoted into serum, plasma, peripheral blood mononuclear cells (PBMC), red blood cells/granulocyte pellet, whole blood, and RNA (totalling 29,863 aliquots). Extensive dataset (700 clinical and socio-demographic variables/participant) enables comprehensive phenotyping. Potential reuse is conditional to ethical approval.
RESUMEN
The laboratory mouse is the workhorse of immunology, used as a model of mammalian immune function, but how well immune responses of laboratory mice reflect those of free-living animals is unknown. Here we comprehensively characterize serological, cellular and functional immune parameters of wild mice and compare them with laboratory mice, finding that wild mouse cellular immune systems are, comparatively, in a highly activated (primed) state. Associations between immune parameters and infection suggest that high level pathogen exposure drives this activation. Moreover, wild mice have a population of highly activated myeloid cells not present in laboratory mice. By contrast, in vitro cytokine responses to pathogen-associated ligands are generally lower in cells from wild mice, probably reflecting the importance of maintaining immune homeostasis in the face of intense antigenic challenge in the wild. These data provide a comprehensive basis for validating (or not) laboratory mice as a useful and relevant immunological model system.
Asunto(s)
Animales de Laboratorio/inmunología , Animales Salvajes/inmunología , Ratones/inmunología , Animales , Proteínas Sanguíneas/metabolismo , Citocinas/biosíntesis , Heces/química , Citometría de Flujo , Haptoglobinas/metabolismo , Homeostasis , Inmunoglobulina A/análisis , Inmunoglobulina E/sangre , Inmunoglobulina G/sangre , Inmunofenotipificación , Activación de Linfocitos , Subgrupos Linfocitarios , Ratones Endogámicos C57BL , Células Mieloides/inmunología , Componente Amiloide P Sérico/metabolismo , Bazo/citología , Bazo/efectos de los fármacos , Bazo/inmunologíaRESUMEN
KCNE3 (MiRP2) forms heteromeric voltage-gated K+ channels with the skeletal muscle-expressed KCNC4 (Kv3.4) α subunit. KCNE3 was the first reported skeletal muscle K+ channel disease gene, but the requirement for KCNE3 in skeletal muscle has been questioned. Here, we confirmed KCNE3 transcript and protein expression in mouse skeletal muscle using Kcne3-/- tissue as a negative control. Whole-transcript microarray analysis (770,317 probes, interrogating 28,853 transcripts) findings were consistent with Kcne3 deletion increasing gastrocnemius oxidative metabolic gene expression and the proportion of type IIa fast-twitch oxidative muscle fibers, which was verified using immunofluorescence. The down-regulated transcript set overlapped with muscle unloading gene expression profiles (≥1.5-fold change; P < 0.05). Gastrocnemius K+ channel α subunit remodeling arising from Kcne3 deletion was highly specific, involving just 3 of 69 α subunit genes probed: known KCNE3 partners KCNC4 and KCNH2 (mERG) were down-regulated, and KCNK4 (TRAAK) was up-regulated (P < 0.05). Functionally, Kcne3-/- mice exhibited abnormal hind-limb clasping upon tail suspension (63% of Kcne3-/- mice ≥10-mo-old vs. 0% age-matched Kcne3+/+ littermates). Whereas 5 of 5 Kcne3+/+ mice exhibited the typical biphasic decline in contractile force with repetitive stimuli of hind-limb muscle, both in vivo and in vitro, this was absent in 6 of 6 Kcne3-/- mice tested. Finally, myoblasts isolated from Kcne3-/- mice exhibit faster-inactivating and smaller sustained outward currents than those from Kcne3+/+ mice. Thus, Kcne3 deletion impairs skeletal muscle function in mice.-King, E. C., Patel, V., Anand, M., Zhao, X., Crump, S. M., Hu, Z., Weisleder, N., Abbott, G. W. Targeted deletion of Kcne3 impairs skeletal muscle function in mice.
Asunto(s)
Regulación de la Expresión Génica/fisiología , Músculo Esquelético/metabolismo , Canales de Potasio con Entrada de Voltaje/metabolismo , Envejecimiento , Animales , Regulación hacia Abajo , Femenino , Ratones , Ratones Noqueados , Contracción Muscular/genética , Contracción Muscular/fisiología , Mioblastos/metabolismo , Canales de Potasio con Entrada de Voltaje/genética , Análisis por Matrices de Proteínas , Transcriptoma , Regulación hacia ArribaRESUMEN
BACKGROUND: Currently, there are no tools to accurately predict tuberculosis relapse. This study aimed to determine whether patients who experience tuberculosis relapse have different immune responses to mycobacteria in vitro than patients who remain cured for 2 years. METHODS: Patients with an initial episode of pulmonary tuberculosis were recruited in South Africa. Diluted blood, collected at diagnosis and after 2 and 4 weeks of treatment, was cultured with live Mycobacterium tuberculosis for 6 days, and cellular RNA was frozen. Gene expression in samples from 10 patients who subsequently experienced relapse, confirmed by strain genotyping, was compared to that in samples from patients who remained cured, using microarrays. RESULTS: At diagnosis, expression of 668 genes was significantly different in samples from patients who experienced relapse, compared with expression in patients who remained successfully cured; these differences persisted for at least 4 weeks. Gene ontology and biological pathways analyses revealed significant upregulation of genes involved in cytotoxic cell-mediated killing. Results were confirmed by real-time quantitative reverse-transcription polymerase chain reaction analysis in a wider patient cohort. CONCLUSIONS: These data show that patients who will subsequently experience relapse exhibit altered immune responses, including excessively robust cytolytic responses to M. tuberculosis in vitro, at the time of diagnosis, compared with patients who will achieve durable cure. Together with microbiological and clinical indices, these differences could be exploited in drug development.
Asunto(s)
Antituberculosos/uso terapéutico , Regulación de la Expresión Génica/inmunología , Recurrencia , Tuberculosis Pulmonar/tratamiento farmacológico , Adulto , Vacuna BCG/inmunología , Biomarcadores , Células Sanguíneas/metabolismo , Femenino , Humanos , Masculino , Persona de Mediana Edad , Mycobacterium bovis , Mycobacterium tuberculosis , ARN Mensajero/genética , ARN Mensajero/metabolismo , Tuberculosis Pulmonar/inmunología , Tuberculosis Pulmonar/prevención & control , Adulto JovenRESUMEN
BACKGROUND: The PD-1 axis is a cell intrinsic immunoregulatory pathway that mediates T cell exhaustion in chronic infection particularly in some viral infections. We hypothesized that PD-1, PD-L1 and PD-L2 would be highly expressed in untreated tuberculosis patients compared to controls due to their chronic infection and would decrease with successful TB treatment. MATERIALS AND METHODS: Untreated tuberculosis patients (n = 26) were recruited at diagnosis and followed up during treatment. Household contacts (n = 24) were recruited to establish baseline differences. Blood gene expression ex vivo was investigated using qRT-PCR. Flow cytometry was performed to establish protein expression patterns. RESULTS: PD-L1 gene expression was found to be elevated in active TB disease; however, this was not observed for PD-1 or PD-L2. The intensive phase of TB treatment was associated with a significant decline in PD-1, PD-L1 and PD-L2 gene expression. PD-1 protein expression on the surface of NK cells, CD8+ and CD4+ T cells was similar in patients with active TB disease compared to controls but declined with successful TB treatment, with the greatest decline occurring on the NK cells followed by CD8+ T cells and then CD4+ T cells. Granzyme B/PD-1 co-expression declined with successful intensive phase treatment. CONCLUSION: Modulation of PD-1/PD-L1 pathway through TB treatment indicates changes in the peripheral T cell response caused by live Mycobacterium tuberculosis (Mtb) followed by the response to dead bacilli, antigen-release and immuno-pathology resolution. The PD-1 axis could be a host drug target for immunomodulatory treatments in the future.
Asunto(s)
Antígeno B7-H1/genética , Células Asesinas Naturales/metabolismo , Mycobacterium tuberculosis , Proteína 2 Ligando de Muerte Celular Programada 1/genética , Receptor de Muerte Celular Programada 1/genética , Subgrupos de Linfocitos T/metabolismo , Tuberculosis/genética , Tuberculosis/metabolismo , Adulto , Anciano , Antituberculosos/uso terapéutico , Antígeno B7-H1/metabolismo , Regulación hacia Abajo , Femenino , Regulación de la Expresión Génica , Humanos , Inmunofenotipificación , Células Asesinas Naturales/inmunología , Recuento de Linfocitos , Masculino , Persona de Mediana Edad , Proteína 2 Ligando de Muerte Celular Programada 1/metabolismo , Receptor de Muerte Celular Programada 1/metabolismo , Subgrupos de Linfocitos T/inmunología , Tuberculosis/tratamiento farmacológico , Tuberculosis/inmunología , Adulto JovenRESUMEN
Cross-species studies enable rapid translational discovery and produce the broadest impact when both mechanism and phenotype are consistent across organisms. We developed a knock-in mouse that biologically recapitulates a common human mutation in the gene for fatty acid amide hydrolase (FAAH) (C385A; rs324420), the primary catabolic enzyme for the endocannabinoid anandamide. This common polymorphism impacts the expression and activity of FAAH, thereby increasing anandamide levels. Here, we show that the genetic knock-in mouse and human variant allele carriers exhibit parallel alterations in biochemisty, neurocircuitry and behaviour. Specifically, there is reduced FAAH expression associated with the variant allele that selectively enhances fronto-amygdala connectivity and fear extinction learning, and decreases anxiety-like behaviours. These results suggest a gain of function in fear regulation and may indicate for whom and for what anxiety symptoms FAAH inhibitors or exposure-based therapies will be most efficacious, bridging an important translational gap between the mouse and human.
Asunto(s)
Amidohidrolasas/genética , Amígdala del Cerebelo/fisiología , Lóbulo Frontal/fisiología , Regulación Enzimológica de la Expresión Génica/genética , Polimorfismo de Nucleótido Simple/genética , Amidohidrolasas/metabolismo , Animales , Western Blotting , Extinción Psicológica/fisiología , Miedo/fisiología , Técnicas de Sustitución del Gen/métodos , Humanos , Procesamiento de Imagen Asistido por Computador , Inmunohistoquímica , Imagen por Resonancia Magnética , Espectrometría de Masas , Ratones , Especificidad de la EspecieRESUMEN
BACKGROUND: Sudden cardiac death (SCD) is the leading global cause of mortality, exhibiting increased incidence in patients with diabetes mellitus. Ion channel gene perturbations provide a well-established ventricular arrhythmogenic substrate for SCD. However, most arrhythmia-susceptibility genes, including the KCNE2 K(+) channel ß subunit, are expressed in multiple tissues, suggesting potential multiplex SCD substrates. METHODS AND RESULTS: Using whole-transcript transcriptomics, we uncovered cardiac angiotensinogen upregulation and remodeling of cardiac angiotensinogen interaction networks in P21 Kcne2(-/-) mouse pups and adrenal remodeling consistent with metabolic syndrome in adult Kcne2(-/-) mice. This led to the discovery that Kcne2 disruption causes multiple acknowledged SCD substrates of extracardiac origin: diabetes mellitus, hypercholesterolemia, hyperkalemia, anemia, and elevated angiotensin II. Kcne2 deletion was also a prerequisite for aging-dependent QT prolongation, ventricular fibrillation and SCD immediately after transient ischemia, and fasting-dependent hypoglycemia, myocardial ischemia, and AV block. CONCLUSIONS: Disruption of a single, widely expressed arrhythmia-susceptibility gene can generate a multisystem syndrome comprising manifold electric and systemic substrates and triggers of SCD. This paradigm is expected to apply to other arrhythmia-susceptibility genes, the majority of which encode ubiquitously expressed ion channel subunits or regulatory proteins.
Asunto(s)
Arritmias Cardíacas/genética , Muerte Súbita Cardíaca/etiología , Síndrome de QT Prolongado/genética , Canales de Potasio con Entrada de Voltaje/genética , Anemia/etiología , Angiotensina II/metabolismo , Angiotensinógeno/genética , Angiotensinógeno/metabolismo , Animales , Arritmias Cardíacas/patología , Diabetes Mellitus/etiología , Dislipidemias/etiología , Redes Reguladoras de Genes , Genotipo , Heterocigoto , Hiperpotasemia/etiología , Isquemia/etiología , Síndrome de QT Prolongado/patología , Ratones , Ratones Noqueados , Canales de Potasio con Entrada de Voltaje/deficiencia , Canales de Potasio con Entrada de Voltaje/metabolismoRESUMEN
Adolescence represents a uniquely sensitive developmental stage in the transition from childhood to adulthood. During this transition, neuronal circuits are particularly susceptible to modification by experience. In addition, adolescence is a stage in which the incidence of anxiety disorders peaks in humans and over 75% of adults with fear-related disorders met diagnostic criteria as children and adolescents. While postnatal critical periods of plasticity for primary sensory processes, such as in the visual system are well established, less is known about potential critical or sensitive periods for fear learning and memory. Here, we review the non-linear developmental aspects of fear learning and memory during a transition period into and out of adolescence. We also review the literature on the non-linear development of GABAergic neurotransmission, a key regulator of critical period plasticity. We provide a model that may inform improved treatment strategies for children and adolescents with fear-related disorders.
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Período Crítico Psicológico , Miedo/fisiología , Aprendizaje/fisiología , Memoria/fisiología , Adolescente , Animales , Trastornos de Ansiedad/etiología , Niño , Condicionamiento Psicológico/fisiología , Extinción Psicológica/fisiología , Humanos , Ratones , Plasticidad Neuronal/fisiología , Ratas , Receptores de GABA/fisiologíaRESUMEN
The transition into and out of adolescence is a unique developmental period during which neuronal circuits are particularly susceptible to modification by experience. Adolescence is associated with an increased incidence of anxiety disorders in humans, and an estimated 75% of adults with fear-related disorders met diagnostic criteria as children and adolescents. Conserved neural circuitry of rodents and humans has facilitated neurodevelopmental studies of behavioral and molecular processes associated with fear learning and memory that lie at the heart of many anxiety disorders. Here, we review the nonlinear developmental aspects of fear learning and memory during a transition period into and out of adolescence and provide a discussion of the molecular mechanisms that may underlie these alterations in behavior. We provide a model that may help to inform novel treatment strategies for children and adolescents with fear-related disorders.
Asunto(s)
Trastornos de Ansiedad/psicología , Desarrollo Infantil , Miedo/psicología , Memoria , Adolescente , Amígdala del Cerebelo/fisiología , Niño , Extinción Psicológica/fisiología , Miedo/fisiología , Humanos , AprendizajeRESUMEN
BACKGROUND: Accurate assessment of treatment efficacy would facilitate clinical trials of new antituberculosis drugs. We hypothesized that early alterations in peripheral immunity could be measured by gene expression profiling in tuberculosis patients undergoing successful conventional combination treatment. METHODS: Ex vivo blood samples from 27 pulmonary tuberculosis patients were assayed at diagnosis and during treatment. RNA was processed and hybridized to Affymetrix GeneChips, to determine expression of over 47,000 transcripts. RESULTS: There were significant ≥ 2-fold changes in expression of >4000 genes during treatment. Rapid, large-scale changes were detected, with down-regulated expression of 1261 genes within the first week, including inflammatory markers such as complement components C1q and C2. This was followed by slower changes in expression of different networks of genes, including a later increase in expression of B-cell markers, transcription factors, and signaling molecules. CONCLUSIONS: The fast initial down-regulation of expression of inflammatory mediators coincided with rapid killing of actively dividing bacilli, whereas slower delayed changes occurred as drugs acted on dormant bacilli and coincided with lung pathology resolution. Measurement of biosignatures during clinical trials of new drugs could be useful predictors of rapid bactericidal or sterilizing drug activity, and would expedite the licensing of new treatment regimens.
Asunto(s)
Antituberculosos/uso terapéutico , Regulación de la Expresión Génica/efectos de los fármacos , Inmunidad Humoral/efectos de los fármacos , Mycobacterium tuberculosis/efectos de los fármacos , Tuberculosis Pulmonar/inmunología , Adolescente , Adulto , Anciano , Linfocitos B/efectos de los fármacos , Estudios de Cohortes , Complemento C1q/efectos de los fármacos , Complemento C2/efectos de los fármacos , Regulación hacia Abajo/efectos de los fármacos , Quimioterapia Combinada , Perfilación de la Expresión Génica , Humanos , Isoniazida/uso terapéutico , Masculino , Persona de Mediana Edad , Análisis de Secuencia por Matrices de Oligonucleótidos , Estudios Prospectivos , Rifampin/uso terapéutico , Tuberculosis Pulmonar/tratamiento farmacológico , Tuberculosis Pulmonar/microbiología , Regulación hacia Arriba/efectos de los fármacos , Adulto JovenRESUMEN
BACKGROUND: Hyperpolarization-activated, cyclic nucleotide-gated (HCN) channels generate the pacemaking current, I(h), which regulates neuronal excitability, burst firing activity, rhythmogenesis, and synaptic integration. The physiological consequence of HCN activation depends on regulation of channel gating by endogenous modulators and stabilization of the channel complex formed by principal and ancillary subunits. KCNE2 is a voltage-gated potassium channel ancillary subunit that also regulates heterologously expressed HCN channels; whether KCNE2 regulates neuronal HCN channel function is unknown. METHODOLOGY/PRINCIPAL FINDINGS: We investigated the effects of Kcne2 gene deletion on I(h) properties and excitability in ventrobasal (VB) and cortical layer 6 pyramidal neurons using brain slices prepared from Kcne2(+/+) and Kcne2(-/-) mice. Kcne2 deletion shifted the voltage-dependence of I(h) activation to more hyperpolarized potentials, slowed gating kinetics, and decreased I(h) density. Kcne2 deletion was associated with a reduction in whole-brain expression of both HCN1 and HCN2 (but not HCN4), although co-immunoprecipitation from whole-brain lysates failed to detect interaction of KCNE2 with HCN1 or 2. Kcne2 deletion also increased input resistance and temporal summation of subthreshold voltage responses; this increased intrinsic excitability enhanced burst firing in response to 4-aminopyridine. Burst duration increased in corticothalamic, but not thalamocortical, neurons, suggesting enhanced cortical excitatory input to the thalamus; such augmented excitability did not result from changes in glutamate release machinery since miniature EPSC frequency was unaltered in Kcne2(-/-) neurons. CONCLUSIONS/SIGNIFICANCE: Loss of KCNE2 leads to downregulation of HCN channel function associated with increased excitability in neurons in the cortico-thalamo-cortical loop. Such findings further our understanding of the normal physiology of brain circuitry critically involved in cognition and have implications for our understanding of various disorders of consciousness.
Asunto(s)
Corteza Cerebral/fisiología , Canales Catiónicos Regulados por Nucleótidos Cíclicos/metabolismo , Eliminación de Gen , Marcación de Gen , Red Nerviosa/fisiología , Canales de Potasio con Entrada de Voltaje/genética , Tálamo/fisiología , 4-Aminopiridina/farmacología , Animales , Corteza Cerebral/efectos de los fármacos , Regulación hacia Abajo/efectos de los fármacos , Femenino , Glutamatos/metabolismo , Canales Regulados por Nucleótidos Cíclicos Activados por Hiperpolarización , Activación del Canal Iónico/efectos de los fármacos , Canales Iónicos/metabolismo , Masculino , Ratones , Ratones Endogámicos C57BL , Red Nerviosa/efectos de los fármacos , Neuronas/efectos de los fármacos , Neuronas/metabolismo , Canales de Potasio/metabolismo , Células Piramidales/efectos de los fármacos , Células Piramidales/metabolismo , Pirimidinas/farmacología , Corteza Somatosensorial/efectos de los fármacos , Corteza Somatosensorial/metabolismo , Tálamo/efectos de los fármacosRESUMEN
Cerebrospinal fluid (CSF) is crucial for normal function and mechanical protection of the CNS. The choroid plexus epithelium (CPe) is primarily responsible for secreting CSF and regulating its composition by mechanisms currently not fully understood. Previously, the heteromeric KCNQ1-KCNE2 K(+) channel was functionally linked to epithelial processes including gastric acid secretion and thyroid hormone biosynthesis. Here, using Kcne2(-/-) tissue as a negative control, we found cerebral expression of KCNE2 to be markedly enriched in the CPe apical membrane, where we also discovered expression of KCNQ1. Targeted Kcne2 gene deletion in C57B6 mice increased CPe outward K(+) current 2-fold. The Kcne2 deletion-enhanced portion of the current was inhibited by XE991 (10 µM) and margatoxin (10 µM) but not by dendrotoxin (100 nM), indicating that it arose from augmentation of KCNQ subfamily and KCNA3 but not KCNA1 K(+) channel activity. Kcne2 deletion in C57B6 mice also altered the polarity of CPe KCNQ1 and KCNA3 trafficking, hyperpolarized the CPe membrane by 9 ± 2 mV, and increased CSF [Cl(-)] by 14% compared with wild-type mice. These findings constitute the first report of CPe dysfunction caused by cation channel gene disruption and suggest that KCNE2 influences blood-CSF anion flux by regulating KCNQ1 and KCNA3 in the CPe.
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Plexo Coroideo/metabolismo , Canal de Potasio KCNQ1/metabolismo , Canal de Potasio Kv1.3/metabolismo , Canales de Potasio con Entrada de Voltaje/metabolismo , Animales , Cloruros/sangre , Cloruros/líquido cefalorraquídeo , Epitelio/metabolismo , Transporte Iónico , Canal de Potasio KCNQ1/química , Canal de Potasio Kv1.3/química , Potenciales de la Membrana , Ratones , Ratones Noqueados , Modelos Biológicos , Canales de Potasio con Entrada de Voltaje/química , Canales de Potasio con Entrada de Voltaje/deficiencia , Canales de Potasio con Entrada de Voltaje/genética , Subunidades de ProteínaRESUMEN
Targeted deletion of the Kcne2 potassium channel ß subunit gene ablates gastric acid secretion and predisposes to gastric neoplasia in mice. Here, we discovered that Kcne2 deletion basolaterally reroutes the Kcnq1 α subunit in vivo in parietal cells (PCs), in which the normally apical location of the Kcnq1-Kcne2 channel facilitates its essential role in gastric acid secretion. Quantitative RT-PCR and Western blotting revealed that Kcne2 deletion remodeled fundic Kcne3 (2.9±0.8-fold mRNA increase, n=10; 5.3±0.4-fold protein increase, n=7) but not Kcne1, 4, or 5, and resulted in basolateral Kcnq1-Kcne3 complex formation in Kcne2(-/-) PCs. Concomitant targeted deletion of Kcne3 (creating Kcne2(-/-)Kcne3(-/-) mice) restored PC apical Kcnq1 localization without Kcne1, 4, or 5 remodeling (assessed by quantitative RT-PCR; n=5-10), indicating Kcne3 actively, basolaterally rerouted Kcnq1 in Kcne2(-/-) PCs. Despite this, Kcne3 deletion exacerbated gastric hyperplasia in Kcne2(-/-) mice, and both hypochlorhydria and hyperplasia in Kcne2(+/-) mice, suggesting that Kcne3 up-regulation was beneficial in Kcne2-depleted PCs. The findings reveal, in vivo, Kcne-dependent α subunit polarized trafficking and the existence and consequences of potassium channel ß subunit remodeling.
Asunto(s)
Regulación de la Expresión Génica/fisiología , Canal de Potasio KCNQ1/metabolismo , Transporte de Proteínas/fisiología , Animales , Femenino , Eliminación de Gen , Hiperplasia/genética , Hiperplasia/patología , Canal de Potasio KCNQ1/genética , Masculino , Ratones , Células Parietales Gástricas/metabolismo , Subunidades de Proteína , Estómago/patología , Gastropatías/genética , Gastropatías/patologíaRESUMEN
Gastric cancer is the second leading cause of cancer death worldwide. Predisposing factors include achlorhydria, Helicobacter pylori infection, oxyntic atrophy and TFF2-expressing metaplasia. In parietal cells, apical potassium channels comprising the KCNQ1 alpha subunit and the KCNE2 beta subunit provide a K(+) efflux current to facilitate gastric acid secretion by the apical H(+)K(+)ATPase. Accordingly, genetic deletion of murine Kcnq1 or Kcne2 impairs gastric acid secretion. Other evidence has suggested a role for KCNE2 in human gastric cancer cell proliferation, independent of its role in gastric acidification. Here, we demonstrate that 1-year-old Kcne2(-/-) mice in a pathogen-free environment all exhibit a severe gastric preneoplastic phenotype comprising gastritis cystica profunda, 6-fold increased stomach mass, increased Ki67 and nuclear Cyclin D1 expression, and TFF2- and cytokeratin 7-expressing metaplasia. Some Kcne2(-/-) mice also exhibited pyloric polypoid adenomas extending into the duodenum, and neoplastic invasion of thin walled vessels in the sub-mucosa. Finally, analysis of human gastric cancer tissue indicated reduced parietal cell KCNE2 expression. Together with previous findings, the results suggest KCNE2 disruption as a possible risk factor for gastric neoplasia.
Asunto(s)
Gastritis/etiología , Gastritis/genética , Canales de Potasio con Entrada de Voltaje/metabolismo , Neoplasias Gástricas/etiología , Neoplasias Gástricas/genética , Animales , Western Blotting , Línea Celular , Línea Celular Tumoral , Ciclina D1/genética , Ciclina D1/metabolismo , Técnica del Anticuerpo Fluorescente , Mucosa Gástrica/metabolismo , Mucosa Gástrica/patología , Eliminación de Gen , ATPasa Intercambiadora de Hidrógeno-Potásio/genética , ATPasa Intercambiadora de Hidrógeno-Potásio/metabolismo , Humanos , Inmunohistoquímica , Canal de Potasio KCNQ1/genética , Canal de Potasio KCNQ1/metabolismo , Antígeno Ki-67/genética , Antígeno Ki-67/metabolismo , Metaplasia/genética , Metaplasia/metabolismo , Ratones , Ratones Mutantes , Péptidos/genética , Péptidos/metabolismo , Canales de Potasio con Entrada de Voltaje/genética , Factor Trefoil-2RESUMEN
Thyroid dysfunction is a global health concern, causing defects including neurodevelopmental disorders, dwarfism and cardiac arrhythmia. Here, we show that the potassium channel subunits KCNQ1 and KCNE2 form a thyroid-stimulating hormone-stimulated, constitutively active, thyrocyte K+ channel required for normal thyroid hormone biosynthesis. Targeted disruption of Kcne2 in mice impaired thyroid iodide accumulation up to eightfold, impaired maternal milk ejection, halved milk tetraiodothyronine (T4) content and halved litter size. Kcne2-deficient mice had hypothyroidism, dwarfism, alopecia, goiter and cardiac abnormalities including hypertrophy, fibrosis, and reduced fractional shortening. The alopecia, dwarfism and cardiac abnormalities were alleviated by triiodothyronine (T3) and T4 administration to pups, by supplementing dams with T(4) before and after they gave birth or by feeding the pups exclusively from Kcne2+/+ dams; conversely, these symptoms were elicited in Kcne2+/+ pups by feeding exclusively from Kcne2-/- dams. These data provide a new potential therapeutic target for thyroid disorders and raise the possibility of an endocrine component to previously identified KCNE2- and KCNQ1-linked human cardiac arrhythmias.