RESUMEN
BACKGROUND & AIMS: Psychological stress is a trigger for the development of irritable bowel syndrome and associated symptoms including abdominal pain. Although irritable bowel syndrome patients show increased activation in the limbic brain, including the amygdala, the underlying molecular and cellular mechanisms regulating visceral nociception in the central nervous system are incompletely understood. In a rodent model of chronic stress, we explored the role of microglia in the central nucleus of the amygdala (CeA) in controlling visceral sensitivity. Microglia are activated by environmental challenges such as stress, and are able to modify neuronal activity via synaptic remodeling and inflammatory cytokine release. Inflammatory gene expression and microglial activity are regulated negatively by nuclear glucocorticoid receptors (GR), which are suppressed by the stress-activated pain mediator p38 mitogen-activated protein kinases (MAPK). METHODS: Fisher-344 male rats were exposed to water avoidance stress (WAS) for 1 hour per day for 7 days. Microglia morphology and the expression of phospho-p38 MAPK and GR were analyzed via immunofluorescence. Microglia-mediated synaptic remodeling was investigated by quantifying the number of postsynaptic density protein 95-positive puncta. Cytokine expression levels in the CeA were assessed via quantitative polymerase chain reaction and a Luminex assay (Bio-Rad, Hercules, CA). Stereotaxic infusion into the CeA of minocycline to inhibit, or fractalkine to activate, microglia was followed by colonic sensitivity measurement via a visceromotor behavioral response to isobaric graded pressures of tonic colorectal distension. RESULTS: WAS induced microglial deramification in the CeA. Moreover, WAS induced a 3-fold increase in the expression of phospho-p38 and decreased the ratio of nuclear GR in the microglia. The number of microglia-engulfed postsynaptic density protein 95-positive puncta in the CeA was increased 3-fold by WAS, while cytokine levels were unchanged. WAS-induced changes in microglial morphology, microglia-mediated synaptic engulfment in the CeA, and visceral hypersensitivity were reversed by minocycline whereas in stress-naïve rats, fractalkine induced microglial deramification and visceral hypersensitivity. CONCLUSIONS: Our data show that chronic stress induces visceral hypersensitivity in male rats and is associated with microglial p38 MAPK activation, GR dysfunction, and neuronal remodeling in the CeA.
Asunto(s)
Núcleo Amigdalino Central/inmunología , Síndrome del Colon Irritable/inmunología , Microglía/inmunología , Estrés Psicológico/complicaciones , Dolor Visceral/inmunología , Animales , Núcleo Amigdalino Central/citología , Núcleo Amigdalino Central/efectos de los fármacos , Núcleo Amigdalino Central/patología , Quimiocina CX3CL1/administración & dosificación , Modelos Animales de Enfermedad , Humanos , Sistema de Señalización de MAP Quinasas/efectos de los fármacos , Sistema de Señalización de MAP Quinasas/inmunología , Masculino , Microglía/efectos de los fármacos , Microglía/patología , Minociclina/administración & dosificación , Plasticidad Neuronal/inmunología , Ratas , Receptores de Glucocorticoides/metabolismo , Técnicas Estereotáxicas , Estrés Psicológico/inmunología , Proteínas Quinasas p38 Activadas por Mitógenos/metabolismoRESUMEN
A promising intervention for Alzheimer's disease (AD) would ideally target key pathological factors that are involved in AD pathogenesis. Soluble factors produced by engrafted mesenchymal stem cells (MSCs) mediate potential therapeutic effects in AD. However, these therapeutic benefits are largely hampered by the limited paracrine capacity of MSCs. In this study, we used adenovirus-mediated gene transduction of bone marrow MSCs to deliver exogenous proteins into the brain of APPswe/PSEN1dE9 (APP/PS1) mice in the early stage of impairment. We observed that engrafted MSCs carrying exogenous (C-X3-C motif) ligand 1 (CX3CL1) alone reduced the production of the inflammatory cytokine TNF-É and improved synapse-related protein expression but not cognitive function. Transplantation of MSCs carrying CX3CL1 and Wnt3a (CX3CL1-Wnt3a-MSC) significantly attenuated the learning and memory impairment when compared with a control group. The improvement of neurobehavioral functions in APP/PS1 mice treated with CX3CL1-Wnt3a-MSC was related to the inhibition of microglial neurotoxicity and promotion of hippocampal neurogenesis. Transplantation of CX3CL1-Wnt3a-MSC also regulated phosphoinositide 3-kinase/activated protein kinase B (PI3K/AKT) signaling to inhibit the activity of glycogen synthase kinase 3 beta (GSK3ß). Taken together, these results indicate that the delivery of exogenous proteins via MSCs can modulate microglial function and enhance neurogenesis, thereby providing new insights into AD intervention.
Asunto(s)
Enfermedad de Alzheimer/terapia , Quimiocina CX3CL1/administración & dosificación , Trasplante de Células Madre Mesenquimatosas , Proteínas/administración & dosificación , Proteína Wnt3A/administración & dosificación , Proteína Wnt3A/metabolismo , Adenoviridae , Enfermedad de Alzheimer/metabolismo , Enfermedad de Alzheimer/fisiopatología , Animales , Células de la Médula Ósea , Quimiocina CX3CL1/metabolismo , Cognición , Modelos Animales de Enfermedad , Células Madre Mesenquimatosas/metabolismo , Ratones Transgénicos , Neurogénesis , Comunicación Paracrina , Transducción Genética , Factor de Necrosis Tumoral alfa/metabolismoRESUMEN
Fractalkine (FKN; CX3CL1) belongs to gamma-chemokine family and binds to CX3CR1 receptors. Currently, the mechanisms involving FKN-induced inflammatory mediators are research targets in an attempt to study immune diseases mechanisms. Besides, FKN seems to modulate inflammation in the nervous system by inducing the secretion of pro-inflammatory mediators such as prostaglandin E2 (PGE2). PGE2 is a classic and important mediator of fever that activates warm-responsive neurons in the anteroventral preoptic region of the hypothalamus (AVPO). Here, we tested the hypothesis that central FKN modulates febrigenic signaling both centrally and peripherally. We performed intracerebroventricular (icv) microinjections of saline (1⯵L) or FKN (doses of 5, 50, 500â¯pg/µL) in rats and measured body temperature (Tb) besides assessing tail skin temperature (Tsk) as a thermoeffector indicator used to calculate the heat loss index (HLI). We also measured the time course changes in AVPO PGE2, besides plasma corticosterone (CORT) and interleukin-6 (IL-6) levels. FKN induced a long lasting febrile response in which the highest dose (500â¯pg/µL) induced a marked rise on Tb that was accompanied by a reduced Tsk and HLI, consequently. FKN increased AVPO PGE2 production in a time-dependent manner besides increasing plasma CORT and IL-6 levels. Our data consistently indicate that FKN increases AVPO PGE2 production and Tb, accompanied by raised plasma IL-6 levels and activation of the hypothalamus-pituitary-adrenal axis.
Asunto(s)
Quimiocina CX3CL1/administración & dosificación , Dinoprostona/metabolismo , Factores Inmunológicos/administración & dosificación , Animales , Temperatura Corporal/efectos de los fármacos , Temperatura Corporal/fisiología , Quimiocina CX3CL1/metabolismo , Corticosterona/sangre , Relación Dosis-Respuesta a Droga , Fiebre/inducido químicamente , Fiebre/inmunología , Infusiones Intraventriculares , Interleucina-6/sangre , Masculino , Neuroinmunomodulación , Ratas WistarRESUMEN
Several lines of evidence indicate that adverse experience in early life may be a triggering factor for pathological inflammatory processes and lead to the development of depression. Fractalkine (CX3CL1), a chemokine, plays an important role not only in the migration, differentiation and proliferation of neuronal and glial cells but also in the regulation of neuronal-microglial signaling and the production of pro-inflammatory factors. In the present study, we examined the impact of a prenatal stress procedure on the expression of fractalkine in the hippocampus and frontal cortex of young and adult male rats. Furthermore, we measured the age-dependent effect of stress during pregnancy on the expression of pro-inflammatory factors IL-1ß, IL-18, TNF-α, IL-6, and CCL2 in both brain structures. Next, to illustrate the link between fractalkine signaling and the behavioral and biochemical changes induced by prenatal stress, adult prenatally stressed offspring were injected intracerebroventricularly (icv) with exogenous fractalkine. We reported that prenatal stress leads to long-lasting deficits in fractalkine signaling and enhanced inflammatory activation. The study demonstrates that icv administration of fractalkine attenuates the behavioural changes evoked by prenatal stress procedure in adult animals. Moreover, fractalkine administration, exhibits anti-inflammatory action, mainly in the frontal cortex of adult prenatally stressed rats. The effect of fractalkine is related to inhibition of NLRP3 inflammasome. However, its action on the other members of NOD-like receptor family (NLR) cannot be excluded. These findings provide new in vivo evidence that the behavioral and inflammatory disturbances observed in adult prenatally stressed rats may be related to long-lasting malfunctions in fractalkine signaling.
Asunto(s)
Conducta Animal , Quimiocina CX3CL1/metabolismo , Hipocampo/metabolismo , Inflamasomas/metabolismo , Proteína con Dominio Pirina 3 de la Familia NLR/metabolismo , Efectos Tardíos de la Exposición Prenatal/metabolismo , Estrés Psicológico/metabolismo , Animales , Conducta Animal/efectos de los fármacos , Quimiocina CX3CL1/administración & dosificación , Quimiocina CX3CL1/genética , Quimiocina CX3CL1/farmacología , Citocinas/genética , Femenino , Hipocampo/crecimiento & desarrollo , Hipocampo/inmunología , Inyecciones Intraventriculares , Masculino , Embarazo , Efectos Tardíos de la Exposición Prenatal/inmunología , Efectos Tardíos de la Exposición Prenatal/psicología , ARN Mensajero/genética , Ratas Sprague-Dawley , Transducción de Señal , Estrés Psicológico/complicaciones , Estrés Psicológico/inmunologíaRESUMEN
OBJECTIVE: Angiogenesis is the formation of new blood vessels through endothelial cell sprouting. This process requires the mitogen-activated protein kinases, signaling molecules that are negatively regulated by the mitogen-activated protein kinase phosphatase-1 (MKP-1). The purpose of this study was to evaluate the role of MKP-1 in neovascularization in vivo and identify associated mechanisms in endothelial cells. APPROACH AND RESULTS: We used murine hindlimb ischemia as a model system to evaluate the role of MKP-1 in angiogenic growth, remodeling, and arteriogenesis in vivo. Genomic deletion of MKP-1 blunted angiogenesis in the distal hindlimb and microvascular arteriogenesis in the proximal hindlimb. In vitro, endothelial MKP-1 depletion/deletion abrogated vascular endothelial growth factor-induced migration and tube formation, and reduced proliferation. These observations establish MKP-1 as a positive mediator of angiogenesis and contrast with the canonical function of MKP-1 as a mitogen-activated protein kinase phosphatase, implying an alternative mechanism for MKP-1-mediated angiogenesis. Cloning and sequencing of MKP-1-bound chromatin identified localization of MKP-1 to exonic DNA of the angiogenic chemokine fractalkine, and MKP-1 depletion reduced histone H3 serine 10 dephosphorylation on this DNA locus and blocked fractalkine expression. In vivo, MKP-1 deletion abrogated ischemia-induced fractalkine expression and macrophage and T-lymphocyte infiltration in distal hindlimbs, whereas fractalkine delivery to ischemic hindlimbs rescued the effect of MKP-1 deletion on neovascular hindlimb recovery. CONCLUSIONS: MKP-1 promoted angiogenic and arteriogenic neovascular growth, potentially through dephosphorylation of histone H3 serine 10 on coding-region DNA to control transcription of angiogenic genes, such as fractalkine. These observations reveal a novel function for MKP-1 and identify MKP-1 as a potential therapeutic target.
Asunto(s)
Fosfatasa 1 de Especificidad Dual/metabolismo , Células Endoteliales/enzimología , Isquemia/enzimología , Músculo Esquelético/irrigación sanguínea , Neovascularización Fisiológica , Animales , Sitios de Unión , Movimiento Celular , Proliferación Celular , Células Cultivadas , Quimiocina CX3CL1/administración & dosificación , Quimiocina CX3CL1/genética , Quimiocina CX3CL1/metabolismo , Modelos Animales de Enfermedad , Fosfatasa 1 de Especificidad Dual/deficiencia , Fosfatasa 1 de Especificidad Dual/genética , Exones , Regulación de la Expresión Génica , Miembro Posterior , Histonas/metabolismo , Células Endoteliales de la Vena Umbilical Humana/enzimología , Humanos , Isquemia/genética , Isquemia/fisiopatología , Isquemia/terapia , Ratones , Ratones Endogámicos C57BL , Ratones Noqueados , Neovascularización Fisiológica/genética , Fosforilación , Interferencia de ARN , Serina , Transducción de Señal , Factores de Tiempo , TransfecciónRESUMEN
Fractalkine (FKN) signaling is involved in mechanical allodynia in the facial skin following trapezius muscle inflammation. Complete Freund's adjuvant (CFA) injection into the trapezius muscle produced mechanical allodynia in the ipsilateral facial skin that was not associated with facial skin inflammation and resulted in FKN but not FKN receptor (CX3CR1) expression, and microglial activation was enhanced in trigeminal spinal subnucleus caudalis (Vc) and upper cervical spinal cord (C1-C2). Intra-cisterna magna anti-CX3CR1 or anti-interleukin (IL)-1ß neutralizing antibody administration decreased the enhanced excitability of Vc and C1-C2 neurons in CFA-injected rats, whereas intra-cisterna magna FKN administration induced microglial activation and mechanical allodynia in the facial skin. IL-1ß expression and p38 mitogen-activated protein kinase phosphorylation were enhanced in activated microglia after CFA injection. The excitability of neurons whose receptive fields was located in the facial skin was significantly enhanced in CFA-injected rats, and the number of cells expressing phosphorylated extracellular signal-regulated kinase (pERK) following noxious mechanical stimulation of the facial skin was significantly increased in Vc and C1-C2. We also observed mechanical allodynia of the trapezius muscle as well as microglial activation and increased pERK expression in C2-C6 after noxious stimulation of the trapezius muscle in facial skin-inflamed rats. These findings suggest that FKN expression was enhanced in Vc and C1-C2 or C2-C6 following trapezius muscle or facial skin inflammation, microglia are activated via FKN signaling, IL-1ß is released from the activated microglia, and the excitability of neurons in Vc and C1-C2 or C2-C6 is enhanced, resulting in the ectopic mechanical allodynia.
Asunto(s)
Quimiocina CX3CL1/metabolismo , Dolor Facial/etiología , Microglía/metabolismo , Músculo Esquelético/patología , Transducción de Señal/fisiología , Animales , Anticuerpos/administración & dosificación , Proteínas de Unión al Calcio/metabolismo , Quimiocina CX3CL1/administración & dosificación , Cisterna Magna/efectos de los fármacos , Cisterna Magna/fisiología , Dermatitis/complicaciones , Dermatitis/tratamiento farmacológico , Modelos Animales de Enfermedad , Dolor Facial/tratamiento farmacológico , Adyuvante de Freund/toxicidad , Hiperalgesia/diagnóstico , Hiperalgesia/etiología , Interleucina-1beta/administración & dosificación , Masculino , Proteínas de Microfilamentos/metabolismo , Microglía/efectos de los fármacos , Actividad Motora/efectos de los fármacos , Músculo Esquelético/efectos de los fármacos , Miositis/inducido químicamente , Miositis/complicaciones , Umbral del Dolor/fisiología , Desempeño Psicomotor/efectos de los fármacos , Ratas , Ratas Sprague-Dawley , Receptores de Interleucina-8A/inmunología , Transducción de Señal/efectos de los fármacosRESUMEN
Here, we demonstrate that the fractalkine (FKN)/CX3CR1 system represents a regulatory mechanism for pancreatic islet ß cell function and insulin secretion. CX3CR1 knockout (KO) mice exhibited a marked defect in glucose and GLP1-stimulated insulin secretion, and this defect was also observed in vitro in isolated islets from CX3CR1 KO mice. In vivo administration of FKN improved glucose tolerance with an increase in insulin secretion. In vitro treatment of islets with FKN increased intracellular Ca(2+) and potentiated insulin secretion in both mouse and human islets. The KO islets exhibited reduced expression of a set of genes necessary for the fully functional, differentiated ß cell state, whereas treatment of wild-type (WT) islets with FKN led to increased expression of these genes. Lastly, expression of FKN in islets was decreased by aging and high-fat diet/obesity, suggesting that decreased FKN/CX3CR1 signaling could be a mechanism underlying ß cell dysfunction in type 2 diabetes.
Asunto(s)
Células Secretoras de Insulina/metabolismo , Insulina/metabolismo , Receptores de Quimiocina/metabolismo , Transducción de Señal , Adulto , Envejecimiento , Animales , Receptor 1 de Quimiocinas CX3C , Cadáver , Quimiocina CX3CL1/administración & dosificación , Quimiocina CX3CL1/metabolismo , Dieta Alta en Grasa , Expresión Génica , Glucosa/metabolismo , Humanos , Hiperglucemia/metabolismo , Secreción de Insulina , Islotes Pancreáticos/citología , Islotes Pancreáticos/patología , Masculino , Ratones , Ratones Endogámicos C57BL , Ratones Noqueados , Persona de Mediana Edad , Receptores de Quimiocina/genéticaRESUMEN
There is increasing evidence that a number of cytokines and their receptors are involved in the processes that lead to the development and maintenance of neuropathic pain states. Here we demonstrate that levels of CX3CR1 (the receptor for the chemokine fractalkine) mRNA in lumbar dorsal root ganglia (DRG) increase 5.8-fold 7 days after sciatic nerve axotomy, and 1.7- and 2.9-fold, 3 and 7 days respectively, after the spared nerve injury (SNI) model of neuropathic pain. In contrast, no significant change in the levels of fractalkine mRNA is apparent in the DRG after axotomy or SNI. The increase in CX3CR1 mRNA is paralleled by a 3.9- and 2.1-fold increase in the number of CX3CR1-positive macrophages in the DRG 7 days after axotomy and SNI, respectively. Expression of CX3CR1 in macrophages is also markedly increased in the sciatic nerve proximal to site of injury, by 25.7-fold after axotomy and 16.2-fold after SNI, 7 days after injury. Intra-neural injection into the sciatic nerve of 400 ng or 100 ng of fractalkine in adult 129OlaHsd mice significantly delayed the development of allodynia for 3 days following SNI. Further, CX3CR1 knockout (KO) mice display an increase in allodynia for three weeks after SNI compared to strain-matched Balb/c controls. Taken together, these results suggest an anti-allodynic role for fractalkine and its receptor in the mouse.
Asunto(s)
Analgésicos/administración & dosificación , Quimiocina CX3CL1/administración & dosificación , Regulación de la Expresión Génica/efectos de los fármacos , Umbral del Dolor/efectos de los fármacos , Ciática/tratamiento farmacológico , Ciática/fisiopatología , Animales , Conducta Animal , Receptor 1 de Quimiocinas CX3C , Modelos Animales de Enfermedad , Relación Dosis-Respuesta a Droga , Ganglios Espinales/efectos de los fármacos , Ganglios Espinales/metabolismo , Regulación de la Expresión Génica/fisiología , Macrófagos/efectos de los fármacos , Macrófagos/metabolismo , Masculino , Ratones , Ratones Endogámicos BALB C , Ratones Noqueados , Dimensión del Dolor , Umbral del Dolor/fisiología , ARN Mensajero/metabolismo , Tiempo de Reacción/efectos de los fármacos , Receptores de Quimiocina/deficiencia , Receptores de Quimiocina/genética , Ciática/genética , Ciática/patología , Médula Espinal/efectos de los fármacos , Médula Espinal/metabolismo , Médula Espinal/patología , Factores de TiempoRESUMEN
The receptor activator of nuclear factor-kappaB ligand (RANKL) is essential for osteoclast differentiation. In this study, we examined the effects of RANKL on chemokine receptor expression in osteoclast precursor cells, RAW264.7 cells. CX3CL1 (also called Fractalkine) receptor, CX3CR1 mRNA expression, was rapidly reduced by treatment with RANKL in contrast to the increased expression of CCR1 and tartrate-resistant acid phosphatase (TRAP). This reduction occurred within 12h and was maintained for 5days during osteoclastogenesis. Inhibitors of phosphatidylinositol 3-kinase (PI3K) and Akt, but not mitogen-activated protein kinases, restored the RANKL-induced reduction of CX3CR1 mRNA. The stability of CX3CR1 mRNA was not changed, suggesting transcriptional repression by RANKL. The down-regulation of CX3CR1 mRNA correlated with the suppression of CX3CL1-induced activation of Akt and ERK as well as chemotaxis. These results suggest a potential role for decreased CX3CL1-CX3CR1 interaction in osteoclastogenesis.