RESUMEN
RATIONALE: Pulmonary arterial hypertension (PH) is a life-threatening condition associated with immune dysregulation and abnormal regulatory T cell (Treg) activity, but it is currently unknown whether and how abnormal Treg function differentially affects males and females. OBJECTIVE: To evaluate whether and how Treg deficiency differentially affects male and female rats in experimental PH. METHODS AND RESULTS: Male and female athymic rnu/rnu rats, lacking Tregs, were treated with the VEGFR2 (vascular endothelial growth factor receptor 2) inhibitor SU5416 or chronic hypoxia and evaluated for PH; some animals underwent Treg immune reconstitution before SU5416 administration. Plasma PGI2 (prostacyclin) levels were measured. Lung and right ventricles were assessed for the expression of the vasoprotective proteins COX-2 (cyclooxygenase 2), PTGIS (prostacyclin synthase), PDL-1 (programmed death ligand 1), and HO-1 (heme oxygenase 1). Inhibitors of these pathways were administered to athymic rats undergoing Treg immune reconstitution. Finally, human cardiac microvascular endothelial cells cocultured with Tregs were evaluated for COX-2, PDL-1, HO-1, and ER (estrogen receptor) expression, and culture supernatants were assayed for PGI2 and IL (interleukin)-10. SU5416-treatment and chronic hypoxia produced more severe PH in female than male athymic rats. Females were distinguished by greater pulmonary inflammation, augmented right ventricular fibrosis, lower plasma PGI2 levels, decreased lung COX-2, PTGIS, HO-1, and PDL-1 expression and reduced right ventricular PDL-1 levels. In both sexes, Treg immune reconstitution protected against PH development and raised levels of plasma PGI2 and cardiopulmonary COX-2, PTGIS, PDL-1, and HO-1. Inhibiting COX-2, HO-1, and PD-1 (programmed death 1)/PDL-1 pathways abrogated Treg protection. In vitro, human Tregs directly upregulated endothelial COX-2, PDL-1, HO-1, ERs and increased supernatant levels of PGI2 and IL-10. CONCLUSIONS: In 2 animal models of PH based on Treg deficiency, females developed more severe PH than males. The data suggest that females are especially reliant on the normal Treg function to counteract the effects of pulmonary vascular injury leading to PH.
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Hipertensión Pulmonar/prevención & control , Factores Sexuales , Linfocitos T Reguladores/fisiología , Inhibidores de la Angiogénesis/farmacología , Animales , Antígeno B7-H1/análisis , Antígeno B7-H1/antagonistas & inhibidores , Antígeno B7-H1/metabolismo , Enfermedad Crónica , Ciclooxigenasa 2/análisis , Ciclooxigenasa 2/metabolismo , Sistema Enzimático del Citocromo P-450/análisis , Sistema Enzimático del Citocromo P-450/metabolismo , Epoprostenol/antagonistas & inhibidores , Epoprostenol/sangre , Epoprostenol/metabolismo , Femenino , Hemo Oxigenasa (Desciclizante)/análisis , Hemo Oxigenasa (Desciclizante)/antagonistas & inhibidores , Hemo Oxigenasa (Desciclizante)/metabolismo , Hipertensión Pulmonar/sangre , Hipertensión Pulmonar/inducido químicamente , Hipertensión Pulmonar/etiología , Hipoxia/complicaciones , Indoles/farmacología , Oxidorreductasas Intramoleculares/análisis , Oxidorreductasas Intramoleculares/antagonistas & inhibidores , Oxidorreductasas Intramoleculares/metabolismo , Pulmón/metabolismo , Masculino , Prostaglandinas I/biosíntesis , Pirroles/farmacología , Ratas , Ratas Desnudas , Receptores de Estrógenos/análisis , Receptores de Estrógenos/antagonistas & inhibidores , Receptores de Estrógenos/metabolismo , Linfocitos T Reguladores/inmunología , Receptor 2 de Factores de Crecimiento Endotelial Vascular/antagonistas & inhibidoresRESUMEN
Cognitive impulsivity is a heritable trait believed to represent the behavior that defines the volition to initiate alcohol drinking. We have previously shown that a neuronal Toll-like receptor 4 (TLR4) signal located in the central amygdala (CeA) and ventral tegmental area (VTA) controls the initiation of binge drinking in alcohol-preferring P rats, and TLR4 expression is upregulated by alcohol-induced corticotropin-releasing factor (CRF) at these sites. However, the function of the TLR4 signal in the nucleus accumbens shell (NAc-shell), a site implicated in the control of reward, drug-seeking behavior and impulsivity and the contribution of other signal-associated genes, are still poorly understood. Here we report that P rats have an innately activated TLR4 signal in NAc-shell neurons that co-express the α2 GABAA receptor subunit and CRF prior to alcohol exposure. This signal is not present in non-alcohol drinking NP rats. The TLR4 signal is sustained by a CRF amplification loop, which includes TLR4-mediated CRF upregulation through PKA/CREB activation and CRF-mediated TLR4 upregulation through the CRF type 1 receptor (CRFR1) and the MAPK/ERK pathway. NAc-shell Infusion of a neurotropic, non-replicating herpes simplex virus vector for TLR4-specific small interfering RNA (pHSVsiTLR4) inhibits TLR4 expression and cognitive impulsivity, implicating the CRF-amplified TLR4 signal in impulsivity regulation.
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Hormona Liberadora de Corticotropina/metabolismo , Conducta Impulsiva/fisiología , Neuronas/metabolismo , Núcleo Accumbens/metabolismo , Receptores de Hormona Liberadora de Corticotropina/metabolismo , Receptores de GABA-A/metabolismo , Receptor Toll-Like 4/metabolismo , Consumo de Bebidas Alcohólicas/metabolismo , Animales , Conducta Animal/fisiología , Comportamiento de Búsqueda de Drogas , Subunidades de Proteína/metabolismo , Ratas , Transducción de Señal/fisiología , Receptor Toll-Like 4/genéticaRESUMEN
Oncolytic virotherapy is a unique cancer therapeutic that encompasses tumour cell lysis through both virus replication and programmed cell death (PCD) pathways. Nonetheless, clinical efficacy is relatively modest, likely related to the immunosuppressive tumour milieu. Our studies use the herpes simplex virus type 2 (HSV-2)-based oncolytic virus ΔPK that has documented anti-tumour activity associated with virus replication, PCD and cancer stem cell lysis. They are designed to examine whether ΔPK-mediated oncolysis includes the ability to reverse the immunosuppressive tumour microenvironment by altering the balance of cytokines directly secreted by the melanoma cells and to define its mechanism. Here, we show that melanoma cells secreted the immunosuppressive cytokine IL-10, and that secretion was inhibited by ΔPK through virus replication and c-Jun N-terminal kinase/c-Jun activation. ΔPK-induced IL-10 inhibition upregulated surface expression of MHC class I chain-related protein A, the ligand for the activating NKG2D receptor expressed on NK- and cytotoxic T-cells. Concomitantly, ΔPK also upregulated the secretion of inflammatory cytokines TNF-α, granulocyte macrophage colony-stimulating factor and IL-1ß through autophagy-mediated activation of Toll-like receptor 2 pathways and pyroptosis, and it inhibited the expression of the negative immune checkpoint regulator cytotoxic T-lymphocyte antigen 4. Pharmacologic inhibition of these processes significantly reduces the oncolytic activity of ΔPK.
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Herpesvirus Humano 2/crecimiento & desarrollo , Tolerancia Inmunológica , Interleucina-10/metabolismo , Melanoma/inmunología , Melanoma/terapia , Viroterapia Oncolítica/métodos , Virus Oncolíticos/crecimiento & desarrollo , Animales , Apoptosis , Autofagia , Línea Celular , Factor Estimulante de Colonias de Granulocitos y Macrófagos/metabolismo , Humanos , Proteínas Quinasas JNK Activadas por Mitógenos/metabolismo , Masculino , Ratones Endogámicos BALB C , Receptor Toll-Like 2/metabolismo , Resultado del Tratamiento , Factor de Necrosis Tumoral alfa/metabolismoRESUMEN
Childhood stress and trauma are associated with substance use disorders in adulthood, but the neurological changes that confer increased vulnerability are largely unknown. In this study, maternal separation (MS) stress, restricted to the pre-weaning period, was used as a model to study mechanisms of protracted effects of childhood stress/traumatic experiences on binge drinking and impulsivity. Using an operant self-administration model of binge drinking and a delay discounting assay to measure impulsive-like behavior, we report that early life stress due to MS facilitated acquisition of binge drinking and impulsivity during adulthood in rats. Previous studies have shown heightened levels of corticotropin releasing factor (CRF) after MS, and here, we add that MS increased expression levels of GABA(A) α2 subunit in central stress circuits. To investigate the precise role of these circuits in regulating impulsivity and binge drinking, the CRF1 receptor antagonist antalarmin and the novel GABA(A) α2 subunit ligand 3-PBC were infused into the central amygdala (CeA) and medial prefrontal cortex (mPFC). Antalarmin and 3-PBC at each site markedly reduced impulsivity and produced profound reductions on binge-motivated alcohol drinking, without altering responding for sucrose. Furthermore, whole-cell patch-clamp studies showed that low concentrations of 3-PBC directly reversed the effect of relatively high concentrations of ethanol on α2ß3γ2 GABA(A) receptors, by a benzodiazepine site-independent mechanism. Together, our data provide strong evidence that maternal separation, i.e. early life stress, is a risk factor for binge drinking, and is linked to impulsivity, another key risk factor for excessive alcohol drinking. We further show that pharmacological manipulation of CRF and GABA receptor signaling is effective to reverse binge drinking and impulsive-like behavior in MS rats. These results provide novel insights into the role of the brain stress systems in the development of impulsivity and excessive alcohol consumption.
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Consumo de Bebidas Alcohólicas/metabolismo , Hormona Liberadora de Corticotropina/metabolismo , Conducta Impulsiva/fisiología , Privación Materna , Receptores de Hormona Liberadora de Corticotropina/metabolismo , Receptores de GABA-A/metabolismo , Estrés Psicológico/metabolismo , Amígdala del Cerebelo/efectos de los fármacos , Amígdala del Cerebelo/metabolismo , Animales , Condicionamiento Operante/efectos de los fármacos , Etanol/administración & dosificación , Femenino , Masculino , Corteza Prefrontal/efectos de los fármacos , Corteza Prefrontal/metabolismo , Pirimidinas/farmacología , Pirroles/farmacología , Ratas , Ratas Sprague-Dawley , Factores de Riesgo , Autoadministración , Vitamina B 12/análogos & derivadosRESUMEN
A growing body of evidence indicates that valproic acid (VPA), a histone deacetylase inhibitor used to treat epilepsy and mood disorders, has histone deacetylase-related and -unrelated neurotoxic activity, the mechanism of which is still poorly understood. We report that VPA induces neuronal cell death through an atypical calpain-dependent necroptosis pathway that initiates with downstream activation of c-Jun N-terminal kinase 1 (JNK1) and increased expression of receptor-interacting protein 1 (RIP-1) and is accompanied by cleavage and mitochondrial release/nuclear translocation of apoptosis-inducing factor, mitochondrial release of Smac/DIABLO, and inhibition of the anti-apoptotic protein X-linked inhibitor of apoptosis (XIAP). Coinciding with apoptosis-inducing factor nuclear translocation, VPA induces phosphorylation of the necroptosis-associated histone H2A family member H2AX, which is known to contribute to lethal DNA degradation. These signals are inhibited in neuronal cells that express constitutively activated MEK/ERK and/or PI3-K/Akt survival pathways, allowing them to resist VPA-induced cell death. The data indicate that VPA has neurotoxic activity and identify a novel calpain-dependent necroptosis pathway that includes JNK1 activation and RIP-1 expression. A growing body of evidence indicates that valproic acid (VPA) has neurotoxic activity, the mechanism of which is still poorly understood. We report, for the first time, that VPA activates a previously unrecognized calpain-dependent necroptosis cascade that initiates with JNK1 activation and involves AIF cleavage/nuclear translocation and H2AX phosphorylation as well as an altered Smac/DIABLO to XIAP balance.
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Calpaína/farmacología , Inhibidores Enzimáticos/farmacología , Regulación de la Expresión Génica/efectos de los fármacos , Neuronas/efectos de los fármacos , Transducción de Señal/efectos de los fármacos , Ácido Valproico/farmacología , Animales , Apoptosis/efectos de los fármacos , Calpaína/metabolismo , Muerte Celular/efectos de los fármacos , Línea Celular , Relación Dosis-Respuesta a Droga , Activación Enzimática/efectos de los fármacos , Humanos , Sistema de Señalización de MAP Quinasas/efectos de los fármacos , Proteína Quinasa 8 Activada por Mitógenos/metabolismo , Mucolipidosis/tratamiento farmacológico , Necrosis/inducido químicamente , Factor de Crecimiento Nervioso/farmacología , Células PC12 , Proteínas Serina-Treonina Quinasas/metabolismo , Ratas , Proteína Serina-Treonina Quinasas de Interacción con Receptores , Factores de TiempoRESUMEN
BACKGROUND: Acute skin erythematous lesions that follow allogeneic hematopoietic stem cell transplantation (HSCT) and are histologically diagnosed as graft-versus-host disease (GVHD) are often associated with reactivation of latent herpes simplex virus (HSV). OBJECTIVE: To further examine the relationship between reactivated HSV and GVHD development. METHODS: We present 3 patients with acute skin GVHD after allogeneic HSCT who were studied prospectively for expression of the HSV antigen Pol, which is involved in HSV-associated erythema multiforme. RESULTS: Pol was expressed in the GVHD lesions but not the pre-HSCT normal skin or peripheral blood mononuclear cells. Lesion severity correlated with the Pol levels but not the histopathologically defined GVHD grade. Lesion development was accompanied by increased numbers of Pol+ circulating/skin-infiltrating CD34+ stem cells and CD1a+ and other dermal dendritic cells. CONCLUSIONS: Subclinical HSV infection of circulating CD34+ cells can contribute to some post-HSCT skin lesions histologically diagnosed as GVHD, with potential preventive and therapeutic implications.
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Antivirales/uso terapéutico , Eritema Multiforme/virología , Enfermedad Injerto contra Huésped , Simplexvirus , Adulto , Femenino , Trasplante de Células Madre Hematopoyéticas/efectos adversos , Humanos , Masculino , Persona de Mediana Edad , Resultado del TratamientoRESUMEN
Binge drinking (blood-alcohol levels ≥ 0.08 g% in a 2-h period), is a significant public health burden in need of improved treatment. Gene therapy may offer beneficial alternatives to current psychosocial and pharmacotherapeutic interventions, but identification of the target genes is a clinical challenge. We report that a GABA(A) α2 siRNA vector (pHSVsiLA2) infused into the central nucleus of the amygdala (CeA) of alcohol-preferring (P) rats caused profound and selective reduction of binge drinking associated with inhibition of α2 expression, decreased GABA(A) receptor density, and inhibition of Toll-like receptor 4 (TLR4). CeA infusion of a TLR4 siRNA vector (pHSVsiLTLR4a) also inhibited binge drinking, but neither vector functioned when infused into the ventral pallidum. Binge drinking was inhibited by a GABA(A) α1 siRNA vector (pHSVsiLA1) infused into the ventral pallidum, unrelated to TLR4. The vectors did not alter sucrose intake and a scrambled siRNA vector was negative. The data indicate that GABA(A) α2-regulated TLR4 expression in the CeA contributes to binge drinking and may be a key early neuroadaptation in excessive drinking.
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Consumo de Bebidas Alcohólicas/metabolismo , Amígdala del Cerebelo/metabolismo , Receptores de GABA-A/metabolismo , Receptor Toll-Like 4/metabolismo , Amígdala del Cerebelo/patología , Animales , Vectores Genéticos , Humanos , ARN Interferente Pequeño/metabolismo , RatasRESUMEN
High genetic heterogeneity is an important characteristic of hepatitis C virus (HCV) that contributes to its ability to establish persistent infection. The hypervariable region 1 (HVR1) that includes the first 27 amino acid residues of the E2 envelope glycoprotein is the most variable region within the HCV polyprotein. HVR1 plays a major role in both HCV cell entry and immune evasion, but the respective contribution of specific amino acid residues is still unclear. Our mutagenesis analyses of HCV pseudoparticles and cell culture-derived HCV using the H77 isolate indicate that five residues at positions 14, 15, and 25-27 mediate binding of the E2 protein to the scavenger receptor class B, type I receptor, and any residue herein is indispensable for HCV cell entry. The region spanning positions 16-24 contains the sole neutralizing epitope and is dispensable for HCV entry, but it is involved in heparan binding. More importantly, this region is necessary for the enhancement of HCV entry by high density lipoprotein and interferes with virus neutralization by E2-neutralizing antibodies. Residues at positions 1-13 are also dispensable for HCV entry, but they can affect HCV infectivity by modulating binding of the envelope protein to scavenger receptor class B, type I. Mutations occurring at this site may confer resistance to HVR1 antibodies. These findings further our understanding about the mechanisms of HCV cell entry and the significance of HVR1 variation in HCV immune evasion. They have major implications for the development of HCV entry inhibitors and prophylactic vaccines.
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Hepacivirus/fisiología , Hepatitis C/inmunología , Evasión Inmune , Proteínas Virales/inmunología , Internalización del Virus , Anticuerpos Neutralizantes/inmunología , Anticuerpos Neutralizantes/farmacología , Anticuerpos Antivirales/inmunología , Anticuerpos Antivirales/farmacología , Antígenos CD36/genética , Antígenos CD36/inmunología , Antígenos CD36/metabolismo , Hepatitis C/genética , Hepatitis C/prevención & control , Humanos , Estructura Terciaria de Proteína , Vacunas contra Hepatitis Viral/genética , Vacunas contra Hepatitis Viral/inmunología , Proteínas Virales/antagonistas & inhibidores , Proteínas Virales/genética , Proteínas Virales/metabolismoRESUMEN
Viruses are implicated in the initiation or flare of graft-versus-host disease (GVHD) by virtue of their ability to activate antigen-presenting dendritic cells (DC). Herpes simplex virus (HSV) infects circulating CD34+ stem cell progenitors, favoring their differentiation into skin homing DC (CD1a+ Langerhans cells) that contribute to the development of an inflammatory skin rash known as HSV-associated erythema multiforme (HAEM). Following on these findings, we conducted a prospective study to examine whether HSV is also associated with GVHD. Skin biopsies and peripheral blood mononuclear cells (PBMC) were collected from 37 consecutive patients on admission before and after allogeneic hematopoietic stem cell transplantation (HSCT) and examined for HSV antigen (Pol) expression and the presence of Pol+CD34+ and Pol+CD1a+ cells. Sixteen patients developed a skin rash that was histopathologically consistent with GVHD (group I), 3 patients had a rash that was not GVHD (group II, EM-like) and 18 patients did not develop any rash after HSCT (group III). Skin biopsies from the group I patients were Pol negative pre-HSCT (baseline) but became Pol+ after the diagnosis of GVHD. The GVHD biopsies also contained Pol+CD34+ and Pol+CD1a+ cells, and these patients had a significant percentage of circulating Pol+CD34+ and Pol+CD1a+ PBMC. By contrast, the group II patients had Pol+ skin cells and Pol+CD34+ circulating PBMC at baseline that decreased post-HSCT. The group III patients had Pol negative skin and very few circulating Pol+CD34+ and Pol+CD1a+ PBMC at baseline that were not significantly changed post-HSCT. The data associate skin GVHD with HSV reactivation during conditioning and its propensity for nonreplicative infection of CD34+ PBMC that induces DC activation. Further studies are needed to better elucidate this association.
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Eritema Multiforme/inmunología , Productos del Gen pol/inmunología , Enfermedad Injerto contra Huésped/inmunología , Trasplante de Células Madre Hematopoyéticas , Herpes Simple/inmunología , Leucocitos Mononucleares/inmunología , Piel/inmunología , Adulto , Antígenos CD1/genética , Antígenos CD1/inmunología , Antígenos CD34/genética , Antígenos CD34/inmunología , Antígenos Virales/genética , Antígenos Virales/inmunología , Diferenciación Celular , Células Dendríticas/inmunología , Células Dendríticas/patología , Células Dendríticas/virología , Eritema Multiforme/etiología , Eritema Multiforme/patología , Eritema Multiforme/virología , Femenino , Expresión Génica , Productos del Gen pol/genética , Enfermedad Injerto contra Huésped/patología , Enfermedad Injerto contra Huésped/virología , Herpes Simple/complicaciones , Herpes Simple/patología , Herpes Simple/virología , Humanos , Células de Langerhans/inmunología , Células de Langerhans/patología , Células de Langerhans/virología , Leucocitos Mononucleares/patología , Leucocitos Mononucleares/virología , Masculino , Persona de Mediana Edad , Estudios Prospectivos , Simplexvirus/inmunología , Piel/patología , Piel/virología , Trasplante HomólogoRESUMEN
BACKGROUND AND AIMS: Muscularis macrophages (MMs) are tissue-resident macrophages in the gut muscularis externa which play a supportive role to the enteric nervous system. We have previously shown that age-dependent MM alterations drive low-grade enteric nervous system inflammation, resulting in neuronal loss and disruption of gut motility. The current studies were designed to identify the MM genetic signature involved in these changes, with particular emphasis on comparison to genes in microglia, the central nervous system macrophage population involved in age-dependent cognitive decline. METHODS: Young (3 months) and old (16-24 months) C57BL/6 mice and human tissue were studied. Immune cells from mouse small intestine, colon, and spinal cord and human colon were dissociated, immunophenotyped by flow cytometry, and examined for gene expression by single-cell RNA sequencing and quantitative real-time PCR. Phagocytosis was assessed by in vivo injections of pHrodo beads (Invitrogen). Macrophage counts were performed by immunostaining of muscularis whole mounts. RESULTS: MMs from young and old mice express homeostatic microglial genes, including Gpr34, C1qc, Trem2, and P2ry12. An MM subpopulation that becomes more abundant with age assumes a geriatric state (GS) phenotype characterized by increased expression of disease-associated microglia genes including Cd9, Clec7a, Itgax (CD11c), Bhlhe40, Lgals3, IL-1ß, and Trem2 and diminished phagocytic activity. Acquisition of the GS phenotype is associated with clearance of α-synuclein aggregates. Human MMs demonstrate a similar age-dependent acquisition of the GS phenotype associated with intracellular α-synuclein accumulation. CONCLUSION: MMs demonstrate age-dependent genetic changes that mirror the microglial disease-associated microglia phenotype and result in functional decline.
RESUMEN
We recently discovered that (3α,5α)3-hydroxypregnan-20-one (allopregnanolone) inhibits pro-inflammatory toll-like receptor (TLR) activation and cytokine/chemokine production in mouse macrophage RAW264.7 cells. The present studies evaluate neurosteroid actions upon TLR activation in human macrophages from male and female healthy donors. Buffy coat leukocytes were obtained from donors at the New York Blood Center (http://nybloodcenter.org/), and peripheral blood mononuclear cells were isolated and cultured to achieve macrophage differentiation. TLR4 and TLR7 were activated by lipopolysaccharide (LPS) or imiquimod in the presence/absence of allopregnanolone or related neurosteroids and pro-inflammatory markers were detected by ELISA or western blotting. Cultured human monocyte-derived-macrophages exhibited typical morphology, a mixed immune profile of both inflammatory and anti-inflammatory markers, with no sex difference at baseline. Allopregnanolone inhibited TLR4 activation in male and female donors, preventing LPS-induced elevations of TNF-α, MCP-1, pCREB and pSTAT1. In contrast, 3α,5α-THDOC and SGE-516 inhibited the TLR4 pathway activation in female, but not male donors. Allopregnanolone completely inhibited TLR7 activation by imiquimod, blocking IL-1-ß, IL-6, pSTAT1 and pIRF7 elevations in females only. 3α,5α-THDOC and SGE-516 partially inhibited TLR7 activation, only in female donors. The results indicate that allopregnanolone inhibits TLR4 and TLR7 activation in cultured human macrophages resulting in diminished cytokine/chemokine production. Allopregnanolone inhibition of TLR4 activation was found in males and females, but inhibition of TLR7 signals exhibited specificity for female donors. 3α,5α-THDOC and SGE-516 inhibited TLR4 and TLR7 pathways only in females. These studies demonstrate anti-inflammatory effects of allopregnanolone in human macrophages for the first time and suggest that inhibition of pro-inflammatory cytokines/chemokines may contribute to its therapeutic actions.
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Activación de Macrófagos , Neuroesteroides , Animales , Quimiocinas/farmacología , Citocinas/metabolismo , Femenino , Humanos , Imiquimod/farmacología , Leucocitos Mononucleares/metabolismo , Lipopolisacáridos/farmacología , Masculino , Ratones , Pregnanolona/farmacología , Transducción de Señal , Receptor Toll-Like 4/metabolismo , Receptor Toll-Like 7 , Receptores Toll-LikeRESUMEN
H11/HspB8 is a functionally distinct small heat shock protein. It causes growth arrest in melanocytes, associated with the inhibition of Cyclin E/Cdk2 and ß-catenin phosphorylation at the transcriptional activity site Ser(552) and is silenced through DNA methylation in 27/35 (77%) melanoma tissues/early cultures. 5-Aza-2'-deoxycytidine (Aza-C) induces melanoma cell death correlated with the levels of H11/HspB8 DNA methylation (p < .001). In line with low/moderate H11/HspB8 methylation, PI3-K inhibition increases Aza-C-induced cell death. Aza-C inhibits the growth of melanoma xenografts related to the levels of H11/HspB8 methylation, and a nonmethylated/non-TAK1 binding H11/HspB8 mutant confers Aza-C resistance. H11/HspB8 is a potential molecular marker for demethylation therapies.
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Antimetabolitos Antineoplásicos/farmacología , Azacitidina/análogos & derivados , Metilación de ADN , Proteínas de Choque Térmico/genética , Melanoma/genética , Proteínas Serina-Treonina Quinasas/genética , Animales , Apoptosis/efectos de los fármacos , Azacitidina/farmacología , Ciclo Celular , Decitabina , Femenino , Proteínas de Choque Térmico/fisiología , Humanos , Melanocitos/fisiología , Melanoma/tratamiento farmacológico , Melanoma/patología , Ratones , Ratones Endogámicos BALB C , Chaperonas Moleculares , Fosfatidilinositol 3-Quinasas/fisiología , Proteínas Serina-Treonina Quinasas/fisiología , Proteínas Proto-Oncogénicas c-akt/fisiología , Ensayos Antitumor por Modelo de XenoinjertoRESUMEN
We have shown that endogenous neurosteroids, including pregnenolone and 3α,5α-THP inhibit toll-like receptor 4 (TLR4) signal activation in mouse macrophages and the brain of alcohol-preferring (P) rat, which exhibits innate TLR4 signal activation. The current studies were designed to examine whether other activated TLR signals are similarly inhibited by 3α,5α-THP. We report that 3α,5α-THP inhibits selective agonist-mediated activation of TLR2 and TLR7, but not TLR3 signaling in the RAW246.7 macrophage cell line. The TLR4 and TLR7 signals are innately activated in the amygdala and NAc from P rat brains and inhibited by 3α,5α-THP. The TLR2 and TLR3 signals are not activated in P rat brain and they are not affected by 3α,5α-THP. Co-immunoprecipitation studies indicate that 3α,5α-THP inhibits the binding of MyD88 with TLR4 or TLR7 in P rat brain, but the levels of TLR4 co-precipitating with TRIF are not altered by 3α,5α-THP treatment. Collectively, the data indicate that 3α,5α-THP inhibits MyD88- but not TRIF-dependent TLR signal activation and the production of pro-inflammatory mediators through its ability to block TLR-MyD88 binding. These results have applicability to many conditions involving pro-inflammatory TLR activation of cytokines, chemokines, and interferons and support the use of 3α,5α-THP as a therapeutic for inflammatory disease.
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Neuroesteroides , Pregnanolona , Animales , Etanol , Ratones , Factor 88 de Diferenciación Mieloide , Pregnenolona , RatasRESUMEN
Hippocampal cultures infected with the DeltaRR vector for the HSV-2 anti-apoptotic gene ICP10PK survive cell death triggered by a wide variety of insults. Survival includes robust protection of uninfected neurons, but the mechanism of this bystander activity is still unclear. Here we report that ICP10PK+ neurons release soluble factors that protect uninfected neurons from NMDA and MPP+-induced apoptosis. Release depends on ICP10PK-mediated activation of the Ras signaling pathways MEK/ERK and PI3-K/Akt, and it was not seen for cultures infected with the ICP10PK negative vector DeltaPK. The released neuroprotective factors include vascular endothelial growth factor (VEGF) and fractalkine, the levels of which were significantly higher in conditioned media from hippocampal cultures infected with DeltaRR (NCM(DeltaRR)) than DeltaPK or phosphate-buffered saline (mock infection). VEGF neutralization inhibited the neuroprotective activity of NCM(DeltaRR), indicating that the VEGF protective function is through neuron-neuron cross-talk. NCM(DeltaRR) also stimulated microglia to release increased levels of IL-10 and decreased levels of TNF-alpha that were protective for uninfected neurons. These release patterns were not seen for microglia given NCM(DeltaRR) in which fractalkine was neutralized, indicating that the fractalkine protective function is through bidirectional neuron-microglia communication. Collectively, the data indicate that DeltaRR is a multiple target strategy to rescue neurons from excitotoxic injury.
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Quimiocina CX3CL1/metabolismo , Quinasas MAP Reguladas por Señal Extracelular/metabolismo , Neuronas/metabolismo , Proteína Oncogénica v-akt/metabolismo , Fosfatidilinositol 3-Quinasas/metabolismo , Proteínas Serina-Treonina Quinasas/metabolismo , Ribonucleótido Reductasas/metabolismo , Factor A de Crecimiento Endotelial Vascular/metabolismo , Análisis de Varianza , Animales , Animales Recién Nacidos , Comunicación Celular/efectos de los fármacos , Comunicación Celular/fisiología , Células Cultivadas , Corteza Cerebral , Chlorocebus aethiops , Medios de Cultivo Condicionados/farmacología , Embrión de Mamíferos , Inhibidores Enzimáticos/farmacología , Ensayo de Inmunoadsorción Enzimática , Vectores Genéticos , Hipocampo/citología , Etiquetado Corte-Fin in Situ/métodos , Interleucina-10/metabolismo , Masculino , Ratones , Microglía/química , Microglía/efectos de los fármacos , Microglía/metabolismo , Mutación , N-Metilaspartato/farmacología , Neuronas/efectos de los fármacos , Técnicas de Cultivo de Órganos , Proteínas Serina-Treonina Quinasas/genética , Proteínas Proto-Oncogénicas c-raf/metabolismo , Ratas , Ribonucleótido Reductasas/genética , Factores de Tiempo , Transfección/métodos , Tubulina (Proteína)/metabolismo , Factor de Necrosis Tumoral alfa/metabolismo , Células VeroRESUMEN
Chemokines such as chemokine (C-C motif) ligand 2 (CCL2) play a role in several behaviors, including anxiety-like behavior, but whether neurons are an important source of CCL2 for behavior and how neuronal CCL2 may work to affect behavior are still debated. When a herpes simplex virus (HSV) vector was used to knockdown CCL2 mRNA in neurons of the central nucleus of the amygdala (CeA) in rats experiencing multiple withdrawals from low dose ethanol, anxiety-like behavior appeared in the social interaction task. To examine this finding further Fractalkine (CX3CL1), a chemokine that is often found to have an opposing function to CCL2 was measured in these rats. Both alcohol withdrawal and CCL2 knockdown increased the levels of the anti-inflammatory protein CX3CL1. The combination of alcohol withdrawal and CCL2 knockdown decreased CX3CL1 and may alter pro-inflammatory/anti-inflammatory balance, and thus highlights the potential importance of CCL2 and CCL2/CX3CL1 balance in anxiety. To find a mechanism by which neuronal chemokines like CCL2 could affect behavior, retrograde tracing with fluorescent nanobeads was done in two brain regions associated with anxiety the bed nucleus of the stria terminalis (BNST) and the ventral periaqueductal gray (VPAG). These studies identified CeA projection neurons to these brain regions that contain CCL2. To demonstrate that CCL2 can be transported via axons to downstream brain regions, the axonal transport blocker, colchicine, was given and 24 h later, the accumulation of CCL2 in CeA neuronal cell bodies was found. Finally, CCL2 in CeA neurons was localized to the synapse using confocal microscopy with enhanced resolution following deconvolution and electron microscopy, which along with the other evidence suggests that CCL2 may be transported down axons in CeA neurons and released from nerve terminals perhaps into brain regions like the BNST and VPAG to affect behaviors such as anxiety. These results suggest that neurons are an important target for chemokine research related to behavior.
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BACKGROUND AND PURPOSE: Toll-like receptors (TLRs) are a family of innate immune system receptors that respond to pathogen-derived and tissue damage-related ligands and are increasingly recognized for their impact on homeostasis and its dysregulation in the nervous system. TLR signaling participates in brain injury and addiction, but its role in the alcohol-seeking behavior, which initiates alcohol drinking, is still poorly understood. In this review, we discuss our findings designed to elucidate the potential contribution of the activated TLR4 signal located in neurons, on impulsivity and the predisposition to initiate alcohol drinking (binge drinking). RESULTS: Our findings indicate that the TLR4 signal is innately activated in neurons from alcohol-preferring subjects, identifying a genetic contribution to the regulation of impulsivity and the alcohol-seeking propensity. Signal activation is through the non-canonical, previously unknown, binding of TLR4 to the α2 subunit of the γ-aminobutyric 2 acid A receptor (GABAAR α2). Activation is sustained by the stress hormone corticotrophin-releasing factor (CRF) and additional still poorly recognized ligand/scaffold proteins. Focus is on the effect of TLR4 signal activation on the balance between pro- and anti-inflammatory chemokines [chemokine (C-C motif) ligand 2 (CCL2)/chemokine (C-X3-C motif) ligand 1 (CX3CL1)] and its effect on binge drinking. CONCLUSION: The results are discussed within the context of current findings on the distinct activation and functions of TLR signals located in neurons, as opposed to immune cells. They indicate that the balance between pro- and anti-inflammatory TLR4 signaling plays a major role in binge drinking. These findings have major impact on future basic and translational research, including the development of potential therapeutic and preventative strategies.
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Consumo Excesivo de Bebidas Alcohólicas/metabolismo , Quimiocina CCL2/metabolismo , Quimiocina CX3CL1/metabolismo , Conducta Impulsiva/fisiología , Neuroinmunomodulación/fisiología , Receptores de GABA-A/metabolismo , Animales , Consumo Excesivo de Bebidas Alcohólicas/inmunología , Quimiocina CCL2/inmunología , Quimiocina CX3CL1/inmunología , Humanos , Receptores de GABA-A/inmunología , Transducción de Señal/fisiología , Receptor Toll-Like 4/inmunología , Receptor Toll-Like 4/metabolismoRESUMEN
The endogenous neurosteroid (3α,5α)3-hydroxypregnan-20-one (3α,5α-THP, allopregnanolone) has protective activity in animal models of alcoholism, depression, traumatic brain injury, schizophrenia, multiple sclerosis, and Alzheimer's disease that is poorly understood. Because these conditions involve proinflammatory signaling through toll-like receptors (TLRs), we examined the effects of 3α,5α-THP, and pregnenolone on TLR4 activation in both the periphery and the central nervous system (CNS). We used monocytes/macrophages (RAW264.7) as a model of peripheral immune signaling and studied innately activated TLR4 in the ventral tegmental area (VTA) of selectively bred alcohol-preferring (P) rats. LPS activated the TLR4 pathway in RAW264.7 cells as evidenced by increased levels of p-TAK1, TRAF6, NF-κB p50, phospho-NF-κB- p65, pCREB, HMGB1, and inflammatory mediators, including MCP-1 and TNFα. Both 3α,5α-THP and pregnenolone (0.5-1.0µM) substantially (~80%) inhibited these effects, indicating pronounced inhibition of TLR4 signaling. The mechanism of inhibition appears to involve blockade of TLR4/MD-2 protein interactions in RAW246.7 cells. In VTA, 3α,5α-THP (15 mg/kg, IP) administration reduced TRAF6 (~20%), CRF (~30%), and MCP-1 (~20%) levels, as well as TLR4 binding to GABAA receptor α2 subunits (~60%) and MyD88 (~40%). The data suggest that inhibition of proinflammatory neuroimmune signaling underlies protective effects of 3α,5α-THP in immune cells and brain, apparently involving blocking of protein-protein interactions that initiate TLR4-dependent signaling. Inhibition of pro-inflammatory TLR4 activation represents a new mechanism of 3α,5α-THP action in the periphery and the brain.
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Inflamación/inmunología , Neuroesteroides/metabolismo , Pregnanolona/metabolismo , Receptor Toll-Like 4/antagonistas & inhibidores , Área Tegmental Ventral/inmunología , Animales , Inflamación/metabolismo , Lipopolisacáridos/inmunología , Masculino , Ratones , Pregnenolona/metabolismo , Mapas de Interacción de Proteínas/inmunología , Células RAW 264.7 , Ratas , Transducción de Señal/inmunología , Receptor Toll-Like 4/metabolismo , Área Tegmental Ventral/metabolismoRESUMEN
Lysosomal enzyme deficiencies comprise a large group of genetic disorders that generally lack effective treatments. A potential treatment approach is to engineer the patient's own hematopoietic system to express high levels of the deficient enzyme, thereby correcting the biochemical defect and halting disease progression. Here, we present an efficient ex vivo genome editing approach using CRISPR-Cas9 that targets the lysosomal enzyme iduronidase to the CCR5 safe harbor locus in human CD34+ hematopoietic stem and progenitor cells. The modified cells secrete supra-endogenous enzyme levels, maintain long-term repopulation and multi-lineage differentiation potential, and can improve biochemical and phenotypic abnormalities in an immunocompromised mouse model of Mucopolysaccharidosis type I. These studies provide support for the development of genome-edited CD34+ hematopoietic stem and progenitor cells as a potential treatment for Mucopolysaccharidosis type I. The safe harbor approach constitutes a flexible platform for the expression of lysosomal enzymes making it applicable to other lysosomal storage disorders.
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Edición Génica/métodos , Genoma Humano , Trasplante de Células Madre Hematopoyéticas/métodos , Células Madre Hematopoyéticas/metabolismo , Iduronidasa/metabolismo , Mucopolisacaridosis I/terapia , Animales , Antígenos CD34/genética , Antígenos CD34/metabolismo , Sistemas CRISPR-Cas , Terapia Genética/métodos , Humanos , Iduronidasa/genética , Ratones , Ratones Endogámicos NOD , Ratones Noqueados , Ratones SCID , Mucopolisacaridosis I/genética , Mucopolisacaridosis I/patología , Células 3T3 NIH , Fenotipo , Receptores CCR5/genética , Receptores CCR5/metabolismo , Trasplante HeterólogoRESUMEN
We have previously shown that intrastriatal injection of Delta RR, the growth-compromised herpes simplex virus type 2 (HSV-2) vector for the antiapoptotic protein ICP10PK, prevents apoptosis caused by the excitotoxin N-methyl-D-aspartate (NMDA) in a mouse model of glutamatergic neuronal cell death (Golembewski et al. [2007] Exp. Neurol. 203:381-393). Because apoptosis regulation is stimulus and cell type specific, our studies were designed to examine the mechanism of Delta RR-mediated neuroprotection in striatal neurons. Organotypic striatal cultures (OSC) that retain much of the synaptic circuitry of the intact striatum were infected with Delta RR or a growth-compromised HSV-2 vector that lacks ICP10PK (Delta PK) and examined for neuroprotection-associated signaling. The mutated ICP10 proteins (p175 and p95) were expressed in 70-80% of neurons from Delta RR- and Delta PK-infected cultures, respectively, as determined by double-immunofluorescent staining with antibodies to ICP10 and NeuN or GAD65. Delta RR- but not Delta PK-treated OSC were protected from NMDA-induced apoptosis, as verified by ethidium homodimer staining, TUNEL, caspase-3 activation, and poly(AD-ribose) polymerase (PARP) cleavage. Neuroprotection was through ICP10PK-mediated activation of the survival pathways MEK/ERK and PI3-K/Akt, up-regulation of the antiapoptotic proteins Bag-1 and Bcl-2, and phosphorylation (inactivation) of the proapoptotic protein Bad. It was blocked by the MEK inhibitor U0126 or the PI3-K inhibitor LY294002, suggesting that either pathway can prevent NMDA-induced apoptosis. The data indicate that Delta RR-delivered ICP10PK stimulates redundant survival pathways that override proapoptotic cascades. Delta RR is a promising gene therapy platform against glutamatergic cell death.
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Apoptosis/fisiología , Terapia Genética/métodos , Proteínas Quinasas Activadas por Mitógenos/metabolismo , Degeneración Nerviosa/prevención & control , Fosfatidilinositol 3-Quinasas/metabolismo , Proteínas Serina-Treonina Quinasas/fisiología , Ribonucleótido Reductasas/fisiología , Animales , Supervivencia Celular/fisiología , Chlorocebus aethiops , Cuerpo Estriado/patología , Agonistas de Aminoácidos Excitadores/toxicidad , Técnica del Anticuerpo Fluorescente , Vectores Genéticos , Herpesvirus Humano 2/genética , Immunoblotting , Etiquetado Corte-Fin in Situ , N-Metilaspartato/toxicidad , Neuronas/patología , Proteínas Serina-Treonina Quinasas/genética , Ratas , Ratas Sprague-Dawley , Ribonucleótido Reductasas/genética , Células VeroRESUMEN
Ischemic brain injury and epilepsy are common neurodegenerative diseases caused by excitotoxicity. Their pathogenesis includes microglial production of inflammatory cytokines. Our studies were designed to examine whether a growth compromised HSV-2 mutant (Delta RR) prevents excitotoxic injury through modulation of microglial responses by the anti-apoptotic HSV-2 protein ICP10PK. EOC2 and EOC20 microglial cells, which are differentially activated, were infected with Delta RR or the ICP10PK deleted virus (Delta PK) and examined for virus-induced neuroprotective activity. Both cell lines were non-permissive for virus growth, but expressed ICP10PK (Delta RR) or the PK deleted ICP10 protein p95 (Delta PK). Conditioned medium (CM) from Delta RR-, but not Delta PK-infected cells prevented N-methyl-D-aspartate (NMDA)-induced apoptosis of primary hippocampal cultures, as determined by TUNEL and caspase-3 activation (76.9 +/- 5.3% neuroprotection). Neuroprotection was associated with inhibition of TNF-alpha and RANTES and production of IL-10. The CM from Delta PK-infected EOC2 and EOC20 cells did not contain IL-10, but it contained TNF-alpha and RANTES. IL-10 neutralization significantly (p < 0.01) decreased, but did not abrogate, the neuroprotective activity of the CM from Delta RR-infected microglial cultures indicating that ICP10PK modulates the neuronal-microglial axis, also through induction of various microglial neuroprotective factors. Rats given Delta RR (but not Delta PK) by intranasal inoculation were protected from kainic acid (KA)-induced seizures and neuronal loss in the CA1 hippocampal fields. Protection was associated with a significant (p < 0.001) increase in the numbers of IL-10+ microglia (CD11b+) as compared to Delta PK-treated animals. Delta RR is a promising vaccination/therapy platform for neurodegeneration through its pro-survival functions in neurons as well as microglia modulation.