RESUMEN
HIV invades the brain during acute infection. Yet, it is unknown whether long-lived infected brain cells release productive virus that can egress from the brain to re-seed peripheral organs. This understanding has significant implication for the brain as a reservoir for HIV and most importantly HIV interplay between the brain and peripheral organs. Given the sheer number of astrocytes in the human brain and their controversial role in HIV infection, we evaluated their infection in vivo and whether HIV infected astrocytes can support HIV egress to peripheral organs. We developed two novel models of chimeric human astrocyte/human peripheral blood mononuclear cells: NOD/scid-IL-2Rgc null (NSG) mice (huAstro/HuPBMCs) whereby we transplanted HIV (non-pseudotyped or VSVg-pseudotyped) infected or uninfected primary human fetal astrocytes (NHAs) or an astrocytoma cell line (U138MG) into the brain of neonate or adult NSG mice and reconstituted the animals with human peripheral blood mononuclear cells (PBMCs). We also transplanted uninfected astrocytes into the brain of NSG mice and reconstituted with infected PBMCs to mimic a biological infection course. As expected, the xenotransplanted astrocytes did not escape/migrate out of the brain and the blood brain barrier (BBB) was intact in this model. We demonstrate that astrocytes support HIV infection in vivo and egress to peripheral organs, at least in part, through trafficking of infected CD4+ T cells out of the brain. Astrocyte-derived HIV egress persists, albeit at low levels, under combination antiretroviral therapy (cART). Egressed HIV evolved with a pattern and rate typical of acute peripheral infection. Lastly, analysis of human cortical or hippocampal brain regions of donors under cART revealed that astrocytes harbor between 0.4-5.2% integrated HIV gag DNA and 2-7% are HIV gag mRNA positive. These studies establish a paradigm shift in the dynamic interaction between the brain and peripheral organs which can inform eradication of HIV reservoirs.
Asunto(s)
Astrocitos , Barrera Hematoencefálica , Infecciones por VIH , VIH-1/metabolismo , Hipocampo , Liberación del Virus , Animales , Antirretrovirales/farmacología , Astrocitos/metabolismo , Astrocitos/patología , Astrocitos/virología , Barrera Hematoencefálica/metabolismo , Barrera Hematoencefálica/patología , Barrera Hematoencefálica/virología , Línea Celular Tumoral , Infecciones por VIH/genética , Infecciones por VIH/metabolismo , Infecciones por VIH/patología , VIH-1/genética , Hipocampo/metabolismo , Hipocampo/patología , Hipocampo/virología , Humanos , Ratones , Ratones Endogámicos NOD , Ratones Noqueados , Ratones SCIDRESUMEN
The variation of macrophage functions suggests the involvement of multiple signalling pathways in fine tuning their differentiation. Macrophages that originate from monocytes in the blood migrate to tissue in response to homeostatic or 'danger' signals and undergo substantial morphological and functional modifications to meet the needs of the dominant signals in the microenvironment. Wnts are secreted glycoproteins that play a significant role in organ and cell differentiation, yet their impact on monocyte differentiation is not clear. In this study, we assessed the role of Wnt1 and Wnt7a on the differentiation of monocytes and the subsequent phenotype and function of monocyte-derived macrophages (MDMs). We show that Wnt7a decreased the expression of CD14, CD11b, CD163 and CD206, whereas Wnt1 had no effect. The Wnt7a effect on CD11b was also observed in the brain and spleen of Wnt7a-/- adult brain mouse tissue and in embryonic Wnt7a-/- tissue. Wnt7a reduced the phagocytic capacity of M-MDMs, decreased interleukin-10 (IL-10) and IL-12 secretion and increased IL-6 secretion. Collectively, these findings demonstrate that Wnt7a generates an MDM phenotype with both pro-inflammatory and alternative MDM cytokine profiles and reduced phagocytic capacity. As such, Wnt7a can have a significant impact on macrophage responses in health and disease.
Asunto(s)
Citocinas/inmunología , Regulación de la Expresión Génica/inmunología , Macrófagos/inmunología , Monocitos/inmunología , Fagocitosis , Proteínas Wnt/inmunología , Animales , Antígenos CD/genética , Antígenos CD/inmunología , Encéfalo/inmunología , Citocinas/genética , Femenino , Humanos , Macrófagos/citología , Masculino , Ratones , Ratones Noqueados , Monocitos/citología , Bazo/inmunología , Proteínas Wnt/genéticaRESUMEN
The role of CD8(+) T cells in HIV control in the brain and the consequences of such control are unclear. Approximately 3% of peripheral CD8(+) T cells dimly express CD4 on their surface. This population is known as CD4(dim)CD8(bright) T cells. We evaluated the role of CD4(dim)CD8(bright) and CD8 single positive T cells in HIV-infected brain using NOD/SCID/IL-2rcγ(-/-) mice reconstituted with human PBMCs (NSG-huPBMC). All three T cell populations (CD4 single positive, CD8 single positive, and CD4(dim)CD8(bright)) were found in NSG-huPBMC mouse brain within 2 wk of infection. Wnts secreted from astrocytes induced CD4(dim)CD8(bright) T cells by 2-fold in vitro. Injection of highly purified CD8 single positive T cells into mouse brain induced CD4(dim)CD8(bright) T cells by 10-fold, which were proliferative and exhibited a terminally differentiated effector memory phenotype. Brain CD4(dim)CD8(bright) T cells from HIV-infected mice exhibited anti-HIV-specific responses, as demonstrated by induction of CD107ab post exposure to HIV peptide-loaded targets. Further, higher frequency of CD4(dim)CD8(bright) T cells (R = -0.62; p ≤ 0.001), but not CD8 single positive T cells (R = -0.24; p ≤ 0.27), negatively correlated with HIV gag mRNA transcripts in HIV-infected NSG-huPBMC brain. Together, these studies indicate that single positive CD8(+) T cells entering the CNS during HIV infection can give rise to CD4(dim)CD8(bright) T cells, likely through a Wnt signaling-dependent manner, and that these cells are associated with potent anti-HIV control in the CNS. Thus, CD4(dim)CD8(bright) T cells are capable of HIV control in the CNS and may offer protection against HIV-associated neurocognitive disorders.
Asunto(s)
Encéfalo/inmunología , Antígenos CD4/metabolismo , Linfocitos T CD8-positivos/inmunología , Infecciones por VIH/inmunología , VIH-1/inmunología , Vía de Señalización Wnt/inmunología , Animales , Astrocitos/metabolismo , Encéfalo/citología , Encéfalo/virología , Antígenos CD8/metabolismo , Linfocitos T CD8-positivos/trasplante , Movimiento Celular/inmunología , Células Cultivadas , Infecciones por VIH/virología , Xenoinjertos , Subunidad gamma Común de Receptores de Interleucina/genética , Leucocitos Mononucleares/inmunología , Leucocitos Mononucleares/trasplante , Activación de Linfocitos/inmunología , Proteína 1 de la Membrana Asociada a los Lisosomas/metabolismo , Proteína 2 de la Membrana Asociada a los Lisosomas/metabolismo , Ratones , Ratones Endogámicos NOD , Ratones Noqueados , Ratones SCID , Bazo/citologíaRESUMEN
BACKGROUND: Neurological disorders have been linked to abnormal excitatory neurotransmission. Perturbations in glutamate cycling can have profound impacts on normal activity, lead to excitotoxicity and neuroinflammation, and induce and/or exacerbate impairments in these diseases. Astrocytes play a key role in excitatory signaling as they both clear glutamate from the synaptic cleft and house enzymes responsible for glutamate conversion to glutamine. However, mechanisms responsible for the regulation of glutamate cycling, including the main astrocytic glutamate transporter excitatory amino acid transporter 2 (EAAT2 or GLT-1 in rodents) and glutamine synthetase (GS) which catalyzes the ATP-dependent reaction of glutamate and ammonia into glutamine, remain largely undefined. METHODS: Gain and loss of function for ß-catenin in human progenitor-derived astrocyte (PDAs) was used to assess EAAT2 and GS levels by PCR, western blot, luciferase reporter assays, and chromatin immunoprecipitation (ChIP). Further, morpholinos were stereotaxically injected into C57BL/6 mice and western blots measured the protein levels of ß-catenin, GLT-1, and GS. RESULTS: ß-Catenin, a transcriptional co-activator and the central mediator of Wnt/ß-catenin signaling pathway, positively regulates EAAT2 and GS at the transcriptional level in PDAs by partnering with T cell factor 1 (TCF-1) and TCF-3, respectively. This pathway is conserved in vivo as the knockdown of ß-catenin in the prefrontal cortex results in reduced GLT-1 and GS expression. CONCLUSIONS: These studies confirm that ß-catenin regulates key proteins responsible for excitatory glutamate neurotransmission in vitro and in vivo and reveal the therapeutic potential of ß-catenin modulation in treating diseases with abnormal glutamatergic neurotransmission and excitotoxicity.
Asunto(s)
Astrocitos/metabolismo , Encéfalo/metabolismo , Ácido Glutámico/metabolismo , beta Catenina/metabolismo , Animales , Encéfalo/efectos de los fármacos , Células Cultivadas , Inmunoprecipitación de Cromatina , Transportador 2 de Aminoácidos Excitadores/genética , Transportador 2 de Aminoácidos Excitadores/metabolismo , Proteína Ácida Fibrilar de la Glía/metabolismo , Humanos , Masculino , Ratones , Morfolinos/farmacología , ARN Mensajero/metabolismo , ARN Interferente Pequeño/genética , ARN Interferente Pequeño/metabolismo , Transducción de Señal/efectos de los fármacos , Transducción de Señal/fisiología , Factores de Transcripción TCF/genética , Factores de Transcripción TCF/metabolismo , Transfección , beta Catenina/genéticaRESUMEN
HIV-mediated neuropathogenesis is a multifaceted process involving several players, including resident brain cells (neurons, astrocytes, and microglia) and infiltrating cells [peripheral blood mononuclear cells (PBMCs)]. We evaluated the dynamic interaction between astrocytes and infiltrating PBMCs as it impacts HIV in the CNS. We demonstrate that human primary-derived astrocytes (PDAs) predominantly secrete Wnt 1, 2b, 3, 5b, and 10b. Wnts are small secreted glycoproteins that initiate either ß-catenin-dependent or independent signal transduction. The Wnt pathway plays a vital role in the regulation of CNS activities including neurogenesis, neurotransmitter release, synaptic plasticity, and memory consolidation. We show that HIV infection of PDAs altered astrocyte Wnt profile by elevating Wnts 2b and 10b. Astrocyte conditioned media (ACM) inhibited HIV replication in PBMCs by 50%. Removal of Wnts from ACM abrogated its ability to suppress HIV replication in PBMCs. Inversely, PBMCs supernatant activated PDAs, as demonstrated by a 10-fold increase in HLA-DR and a 5-fold increase in IFNγ expression, and enhanced astrocyte susceptibility to HIV by 2-fold, which was mediated by IFNγ in a Stat-3-dependent manner. Collectively, these data demonstrate a dynamic interaction between astrocytes and PBMCs, whereby astrocyte-secreted Wnts exert an anti-HIV effect on infected PBMCs and PBMCs, in turn, secrete IFNγ that enhance astrocyte susceptibility to productive HIV infection and mediate their activation.
Asunto(s)
Complejo SIDA Demencia/fisiopatología , Astrocitos/fisiología , Leucocitos Mononucleares/fisiología , Complejo SIDA Demencia/virología , Astrocitos/virología , Linfocitos T CD8-positivos/metabolismo , Células Cultivadas , Medios de Cultivo Condicionados , VIH/fisiología , Antígenos HLA-DR/metabolismo , Humanos , Interferón gamma/metabolismo , Leucocitos Mononucleares/virología , Factor de Transcripción STAT3/metabolismo , Proteínas Wnt/metabolismoRESUMEN
System x(c)(-) represents an intriguing target in attempts to understand the pathological states of the central nervous system. Also called a cystine-glutamate antiporter, system x(c)(-) typically functions by exchanging one molecule of extracellular cystine for one molecule of intracellular glutamate. Nonvesicular glutamate released during cystine-glutamate exchange activates extrasynaptic glutamate receptors in a manner that shapes synaptic activity and plasticity. These findings contribute to the intriguing possibility that extracellular glutamate is regulated by a complex network of release and reuptake mechanisms, many of which are unique to glutamate and rarely depicted in models of excitatory signaling. Because system x(c)(-) is often expressed on non-neuronal cells, the study of cystine-glutamate exchange may advance the emerging viewpoint that glia are active contributors to information processing in the brain. It is noteworthy that system x(c)(-) is at the interface between excitatory signaling and oxidative stress, because the uptake of cystine that results from cystine-glutamate exchange is critical in maintaining the levels of glutathione, a critical antioxidant. As a result of these dual functions, system x(c)(-) has been implicated in a wide array of central nervous system diseases ranging from addiction to neurodegenerative disorders to schizophrenia. In the current review, we briefly discuss the major cellular components that regulate glutamate homeostasis, including glutamate release by system x(c)(-). This is followed by an in-depth discussion of system x(c)(-) as it relates to glutamate release, cystine transport, and glutathione synthesis. Finally, the role of system x(c)(-) is surveyed across a number of psychiatric and neurodegenerative disorders.
Asunto(s)
Sistema de Transporte de Aminoácidos y+/metabolismo , Sistema Nervioso Central , Cistina/metabolismo , Ácido Glutámico/metabolismo , Sinapsis , Animales , Transporte Biológico , Sistema Nervioso Central/metabolismo , Sistema Nervioso Central/patología , Glutatión/biosíntesis , Humanos , Trastornos Mentales/metabolismo , Trastornos Mentales/patología , Enfermedades Neurodegenerativas/metabolismo , Enfermedades Neurodegenerativas/patología , Síndromes de Neurotoxicidad/metabolismo , Síndromes de Neurotoxicidad/patología , Transducción de Señal , Sinapsis/metabolismo , Sinapsis/patologíaRESUMEN
Factors that result in augmented reinstatement, including increased withdrawal period duration and high levels of cocaine consumption, may provide insight into relapse vulnerability. The neural basis of augmented reinstatement may arise from more pronounced changes in plasticity required for reinstatement and/or the emergence of plasticity expressed only during a specific withdrawal period or under specific intake conditions. In this study, we examined the impact of withdrawal period duration and cocaine intake on the magnitude of cocaine-primed reinstatement and extracellular glutamate in the nucleus accumbens, which has been shown to be required for cocaine-primed reinstatement. Rats were assigned to self-administer under conditions resulting in low (2 hours/day; 0.5 mg/kg/infusion, IV) or high (6 hours/day; 1.0 mg/kg/infusion, IV) levels of cocaine intake. After 1, 21 or 60 days of withdrawal, drug seeking and extracellular glutamate levels in the nucleus accumbens were measured before and after a cocaine injection. Cocaine-reinstated lever pressing and elevated extracellular glutamate at every withdrawal time point tested, which is consistent with the conclusion that increased glutamatergic signaling in the nucleus accumbens, is required for cocaine-induced reinstatement. Interestingly, high-intake rats exhibited augmented reinstatement at every time point tested, yet failed to exhibit higher levels of cocaine-induced increases in extracellular glutamate relative to low-intake rats. Our current data indicate that augmented reinstatement in high-intake rats is not due to relative differences in extracellular levels of glutamate in the nucleus accumbens, but rather may stem from intake-dependent plasticity.
Asunto(s)
Conducta Animal/efectos de los fármacos , Trastornos Relacionados con Cocaína/metabolismo , Trastornos Relacionados con Cocaína/psicología , Cocaína/farmacología , Plasticidad Neuronal/efectos de los fármacos , Animales , Cocaína/administración & dosificación , Cocaína/metabolismo , Modelos Animales de Enfermedad , Relación Dosis-Respuesta a Droga , Comportamiento de Búsqueda de Drogas/efectos de los fármacos , Ácido Glutámico/metabolismo , Masculino , Núcleo Accumbens/efectos de los fármacos , Núcleo Accumbens/metabolismo , Ratas , Ratas Sprague-Dawley , Recurrencia , Autoadministración , Síndrome de Abstinencia a Sustancias/metabolismo , Síndrome de Abstinencia a Sustancias/psicología , Factores de TiempoRESUMEN
RATIONALE: Gaps in our understanding of glutamatergic signaling may be key obstacles in accurately modeling complex CNS diseases. System xc (-) is an example of a poorly understood component of glutamate homeostasis that has the potential to contribute to CNS diseases. OBJECTIVES: This study aims to determine whether system xc (-) contributes to behaviors used to model features of CNS disease states. METHODS: In situ hybridization was used to map mRNA expression of xCT throughout the brain. Microdialysis in the prefrontal cortex was used to sample extracellular glutamate levels; HPLC was used to measure extracellular glutamate and tissue glutathione concentrations. Acute administration of sulfasalazine (8-16 mg/kg, IP) was used to decrease system xc (-) activity. Behavior was measured using attentional set shifting, elevated plus maze, open-field maze, Porsolt swim test, and social interaction paradigm. RESULTS: The expression of xCT mRNA was detected throughout the brain, with high expression in several structures including the basolateral amygdala and prefrontal cortex. Doses of sulfasalazine that produced a reduction in extracellular glutamate levels were identified and subsequently used in the behavioral experiments. Sulfasalazine impaired performance in attentional set shifting and reduced the amount of time spent in an open arm of an elevated plus maze and the center of an open-field maze without altering behavior in a Porsolt swim test, total distance moved in an open-field maze, or social interaction. CONCLUSIONS: The widespread distribution of system xc (-) and involvement in a growing list of behaviors suggests that this form of nonvesicular glutamate release is a key component of excitatory signaling.
Asunto(s)
Conducta Animal/fisiología , Encéfalo/metabolismo , Ácido Glutámico/metabolismo , Animales , Atención/efectos de los fármacos , Atención/fisiología , Conducta Animal/efectos de los fármacos , Encéfalo/efectos de los fármacos , Glutatión/metabolismo , Hibridación in Situ , Relaciones Interpersonales , Masculino , Microdiálisis , Actividad Motora/efectos de los fármacos , Actividad Motora/fisiología , ARN Mensajero/metabolismo , Ratas , Ratas Sprague-Dawley , Sulfasalazina/farmacologíaRESUMEN
HIV infection is a risk factor for the tumorigenesis including non-AIDS-defining cancers such as those of the gastrointestinal tract. However, the mechanisms underlying such cancer outgrowth are still unknown. Furthermore, combined HIV/cancer studies are difficult to evaluate using primate models or in the clinical patient setting. To understand the mechanisms of tumor outgrowth in the context of HIV infection, we adopted a humanized mouse model permissive to infection and cancer as well as an in vivo humanized mouse challenge with colon cancer in the context of HIV infection. Immunodeficient NOD SCID IL-2R(-/-) mice were immunologically reconstituted by adoptive transfer of 10(7) HIV-negative donor peripheral blood leukocytes and challenged with 10(6) HCT116 human colon cancer cells. A group of mice was treated with antiretroviral therapy. Tumor microenvironment and epithelial tissues in the context of HIV infection were analyzed using immunohistochemistry. We demonstrate that HIV-infected humanized mice develop significantly larger tumors than uninfected mice (p<0.05). Epithelial cell proliferation in HIV-infected mice is significantly enhanced in comparison to proliferation in uninfected mice (p<0.01). Moreover, the activation of ß-catenin, an important step in intestinal epithelial cell proliferation and tumorigenesis, is elevated in the tumors of HIV-infected mice (p<0.0001). Importantly, antiretroviral therapy reverses these pathological processes independently of CD4(+) T cell return. These findings model the ability of HIV infection to result in tumor outgrowth that is evident in HIV-positive patients and lend insight into previously unrecognized mechanisms that may underlie this pathology.
Asunto(s)
Neoplasias Gastrointestinales/inmunología , Neoplasias Gastrointestinales/patología , Infecciones por VIH/inmunología , VIH-1/inmunología , Leucocitos/inmunología , Traslado Adoptivo , Animales , Antirretrovirales/uso terapéutico , Linfocitos T CD4-Positivos/inmunología , Carcinogénesis , Línea Celular Tumoral , Proliferación Celular , Modelos Animales de Enfermedad , Neoplasias Gastrointestinales/virología , Células HCT116 , Infecciones por VIH/tratamiento farmacológico , Infecciones por VIH/virología , Humanos , Transfusión de Leucocitos , Ratones , Ratones Endogámicos NOD , Ratones Noqueados , Ratones SCID , Neoplasias Experimentales/inmunología , Neoplasias Experimentales/patología , Receptores de Interleucina-2/genética , beta Catenina/biosíntesisRESUMEN
RATIONALE: Aspects of schizophrenia, including deficits in sensorimotor gating, have been linked to glutamate dysfunction and/or oxidative stress in the prefrontal cortex. System xc(-), a cystine-glutamate antiporter, is a poorly understood mechanism that contributes to both cellular antioxidant capacity and glutamate homeostasis. OBJECTIVES: Our goal was to determine whether increased system xc(-) activity within the prefrontal cortex would normalize a rodent measure of sensorimotor gating. METHODS: In situ hybridization was used to map messenger RNA (mRNA) expression of xCT, the active subunit of system xc(-), in the prefrontal cortex. Prepulse inhibition was used to measure sensorimotor gating; deficits in prepulse inhibition were produced using phencyclidine (0.3-3 mg/kg, sc). N-Acetylcysteine (10-100 µM) and the system xc(-) inhibitor (S)-4-carboxyphenylglycine (CPG, 0.5 µM) were used to increase and decrease system xc(-) activity, respectively. The uptake of (14)C-cystine into tissue punches obtained from the prefrontal cortex was used to assay system xc(-) activity. RESULTS: The expression of xCT mRNA in the prefrontal cortex was most prominent in a lateral band spanning primarily the prelimbic cortex. Although phencyclidine did not alter the uptake of (14)C-cystine in prefrontal cortical tissue punches, intraprefrontal cortical infusion of N-acetylcysteine (10-100 µM) significantly reduced phencyclidine- (1.5 mg/kg, sc) induced deficits in prepulse inhibition. N-Acetylcysteine was without effect when coinfused with CPG (0.5 µM), indicating an involvement of system xc(-). CONCLUSIONS: These results indicate that phencyclidine disrupts sensorimotor gating through system xc(-) independent mechanisms, but that increasing cystine-glutamate exchange in the prefrontal cortex is sufficient to reduce behavioral deficits produced by phencyclidine.