RESUMO
BACKGROUND/AIMS: Neuroendocrine dysregulation has been associated with rheumatoid arthritis (RA). Tyrosine hydroxylase (TH), a rate-limiting enzyme for synthesis of neuroendocrine hormones such as epinephrine, is also expressed in T lymphocytes and regulates balance between helper T (Th) 17 cells and regulatory T (Treg) cells. Herein, we aimed to show that TH expression in joints alleviates joint inflammation and Th17/Treg imbalance in collagen-induced arthritis (CIA), an animal model of RA, and these effects may be implemented by the mechanism of epinephrine action on α1-adrenoreceptor (α1-AR) in T cells. METHODS: CIA was prepared by intradermal injection of collagen type II in tail base of DBA1/J mice. On the 33rd day post-immunization, lentiviral vectors encoding TH or TH shRNA were injected into ankle joints of CIA mice. Limb inflammation of the mice was assessed beginning from day 21 until day 69 post-immunization by measurement of limb swelling, erythema and rigidity. Th17 and Treg differentiation and function in ankle joints were assessed on day 69 post-immunization by test of the expression of Th17 transcriptional factor ROR-γt and the levels of proinflammatory cytokines interleukin (IL)-17 and IL-22 as well as the expression of Treg transcriptional factor Foxp3 and the levels of antiinflammatory cytokines transforming growth factor (TGF)-ß1 and IL-10. T cells were obtained from the spleen of mice that had been immunized with collagen type II 41 day earlier and treated with epinephrine or α1-AR agonist phenylephrine in vitro. Flow cytometry was used to analyze the percentages of CD25-IL-17+ cells and CD25+Foxp3+ cells in CD4+ T cells. RESULTS: TH gene overexpression in ankle joints of CIA mice reduced limb inflammation and Th17-related transcription factor expression and inflammatory cytokine production but increased Treg-related antiinflammatory cytokine production in the joints. In contrast, TH gene silence in ankle joints of CIA mice enhanced limb inflammation and Th17 cell activity but decreased Treg cell function in the joints. Epinephrine upregulated α1-AR expression in T cells derived from CIA mice. Both epinephrine and phenylephrine reduced CIA-induced Th17 transcription factor expression and inflammatory cytokine production but enhanced Treg antiinflammatory cytokine production in vitro. CONCLUSION: Upregulating TH expression in joints alleviates joint inflammation and Th17/Treg imbalance in CIA at least partially by enhancing epinephrine action on α1-AR in T cells.
Assuntos
Artrite Experimental , Células Th17 , Animais , Inflamação , Camundongos , Linfócitos T Reguladores , Tirosina 3-Mono-OxigenaseRESUMO
Parkinson's disease (PD) is a chronic neurodegenerative disease. Recently, neuroinflammation driven by CD4+ T cells has been involved in PD pathophysiology. Human and murine lymphocytes express all the five subtypes of dopamine receptors (DRs), DRD1 to DRD5. However, roles of DRs particularly DRD2 expressed on CD4+ T cells in PD remain elucidated. Global Drd1- or Drd2-knockout (Drd1-/- or Drd2-/-) mice or CD4+ T cell-specific Drd2-knockout (Drd2fl/fl/CD4Cre) mice were intraperitoneally injected with 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine (MPTP) to induce PD with the different mutants. On the 7th day following MPTP injection, mice were assessed for dopaminergic neurodegeneration, locomotor impairments, microglial activation, as well as CD4+ T-cell differentiation and function. Furthermore, in vitro CD4+ T cells were exposed to DRD2 agonist and antagonist and then differentiation and function of the cells were determined. MPTP induced dopaminergic neuronal loss in the nigrostriatal system, motor coordinative and behavioral impairments, microglial activation, and CD4+ T-cell polarization to pro-inflammatory T-helper (Th)1 and Th17 phenotypes. Importantly, either Drd2-/- or Drd2fl/fl/CD4Cre mice manifested more severe dopaminergic neurodegeneration, motor deficits, microglial activation, and CD4+ T-cell bias towards Th1 and Th17 phenotypes in response to MPTP, but Drd1-/- did not further alter MPTP intoxication. DRD2 agonist sumanirole inhibited shift of CD4+ T cells obtained from MPTP-intoxicated mice to Th1 and Th17 phenotypes and DRD2 antagonist L-741,626 reversed sumanirole effects. These findings suggest that DRD2 expressed on CD4+ T cells is protective against neuroinflammation and neurodegeneration in PD. Thus, developing a therapeutic strategy of stimulating DRD2 may be promising for mitigation of PD.
Assuntos
Doenças Neurodegenerativas , Doença de Parkinson , 1-Metil-4-Fenil-1,2,3,6-Tetra-Hidropiridina , Animais , Modelos Animais de Doenças , Neurônios Dopaminérgicos , Camundongos , Camundongos Endogâmicos C57BL , Doenças Neuroinflamatórias , Receptores de Dopamina D2 , Receptores de Dopamina D5 , Células Th17RESUMO
Neuroinflammation has been involved in pathogenesis of Parkinson's disease (PD), a chronic neurodegenerative disease characterized neuropathologically by progressive dopaminergic neuronal loss in the substantia nigra (SN). We recently have shown that helper T (Th)17 cells facilitate dopaminergic neuronal loss in vitro. Herein, we demonstrated that interleukin (IL)-17A, a proinflammatory cytokine produced mainly by Th17 cells, contributed to PD pathogenesis depending on microglia. Mouse and rat models for PD were prepared by intraperitoneal injection of 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine (MPTP) or striatal injection of 1-methyl-4-phenylpyridinium (MPP+), respectively. Both in MPTP-treated mice and MPP+-treated rats, blood-brain barrier (BBB) was disrupted and IL-17A level increased in the SN but not in cortex. Effector T (Teff) cells that were adoptively transferred via tail veins infiltrated into the brain of PD mice but not into that of normal mice. The Teff cell transfer aggravated nigrostriatal dopaminergic neurodegeneration, microglial activation and motor impairment. Contrarily, IL-17A deficiency alleviated BBB disruption, dopaminergic neurodegeneration, microglial activation and motor impairment. Anti-IL-17A-neutralizing antibody that was injected into lateral cerebral ventricle in PD rats ameliorated the manifestations mentioned above. IL-17A activated microglia but did not directly affect dopaminergic neuronal survival in vitro. IL-17A exacerbated dopaminergic neuronal loss only in the presence of microglia, and silencing IL-17A receptor gene in microglia abolished the IL-17A effect. IL-17A-treated microglial medium that contained higher concentration of tumor necrosis factor (TNF)-α facilitated dopaminergic neuronal death. Further, TNF-α-neutralizing antibody attenuated MPP+-induced neurotoxicity. The findings suggest that IL-17A accelerates neurodegeneration in PD depending on microglial activation and at least partly TNF-α release.
Assuntos
Interleucina-17/imunologia , Microglia/imunologia , Doença de Parkinson/imunologia , 1-Metil-4-fenilpiridínio/farmacologia , Animais , Morte Celular/imunologia , Corpo Estriado/imunologia , Modelos Animais de Doenças , Dopamina/imunologia , Neurônios Dopaminérgicos/imunologia , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Degeneração Neural/imunologia , Degeneração Neural/patologia , Doenças Neurodegenerativas/imunologia , Doenças Neurodegenerativas/patologia , Neuroimunomodulação/imunologia , Ratos , Ratos Sprague-Dawley , Substância Negra/imunologia , Células Th17/imunologia , Fator de Necrose Tumoral alfa/metabolismo , Tirosina 3-Mono-Oxigenase/metabolismoRESUMO
Neuroinflammation is principally linked to glial function and has been demonstrated to participate in the pathogenesis of Alzheimer's disease, a neurodegenerative disorder characterized by beta-amyloid ccumulation and neurotransmission disruption. Previous findings suggest acetylcholine exerts anti-inflammatory and neuroprotective properties in several neurodegenerative disorders. However, the underlying mechanisms remain elusive. Here evaluation of the influence of acetylcholine on neuroinflammation and neurodegeneration in Alzheimer's disease is reported and further neuroprotective mechanisms are investigated. Investigation of microglia in lipopolysaccharide-induced hippocampal neuronal toxicity employed α7nAChR gene silencing and demonstrated that both the anti-inflammatory and neuroprotective effects of acetylcholine rely on α7nAChR pathways. As expected, in neuron-microglia co-cultures lipopolysaccharide induced an increase in expression of pro-inflammatory factors, including inducible nitric oxide synthase, interleukin-1α, and tumor necrosis factor-α, and decreased expression of neurotrophic factors such as insulin-like growth factor-1, and neuronal apoptosis. Acetylcholine protects against lipopolysaccharide-elicited neuronal injury by inhibiting the microglial inflammatory response and promoting microglial neurotrophic factor production via the action of α7nAChR on microglia. These findings establish that ACh activates α7nAChR in microglia, which in turn protects hippocampal neurons.
Assuntos
Acetilcolina/metabolismo , Hipocampo/metabolismo , Inflamação/metabolismo , Microglia/metabolismo , Neurônios/metabolismo , Neuroproteção/fisiologia , Animais , Apoptose/fisiologia , Técnicas de Cocultura , Escherichia coli , Lipopolissacarídeos , Cultura Primária de Células , Ratos Sprague-Dawley , Receptor Nicotínico de Acetilcolina alfa7/metabolismoRESUMO
BACKGROUND Norepinephrine (NE), a neurotransmitter released from the sympathetic nerves, has been shown to be involved in rheumatoid arthritis (RA). However, its role in the sympathetic nervous system in RA is divergent. Herein, we demonstrate that the sympathetic neurotransmitter NE exerts an anti-inflammatory effect in collagen-induced arthritis (CIA), a mouse model of RA, by inhibiting Th17 cell differentiation and function via ß2-adrenergic receptor (ß2-AR) signaling. MATERIAL AND METHODS CIA was prepared by intradermal injection of collagen type II in the tail base of DBA1/J mice. On the 41st day post-immunization, the mice were used as CIA models. CD4+ T cells from the spleen were purified using magnetic cell sorting and activated with anti-CD3 anti-CD28 antibodies. Th17 cells were polarized from the CD4+ T cells using various antibodies and cytokines. RESULTS Co-expression of CD4 and ß2-AR was observed in spleens of both intact and CIA mice. The ß2-AR expression in the ankle and spleen was downregulated in CIA mice. CIA induced increases in production of interleukin (IL)-17 and IL-22, CD25-IL-17+ cell percentage, and ROR-γt expression in CD4+ T cells. Importantly, NE reduced the CIA-induced CD4+ T cell shift towards Th17 phenotype, and the ß2-AR antagonist ICI118551 blocked the NE effect. Moreover, the ß2-AR agonist terbutaline (Terb) inhibited CIA-induced CD4+ T cell proliferation and shift towards Th17 phenotype, and the protein kinase A (PKA) inhibitor H-89 abolished the agonist effect. Terb also reduced CIA-induced Th17 enhancement, and H-89 impaired the Terb effect. CONCLUSIONS NE inhibits Th17 cell differentiation and function in CIA condition by activation of ß2-AR/PKA signaling.
Assuntos
Artrite Reumatoide/imunologia , Artrite Reumatoide/metabolismo , Norepinefrina/uso terapêutico , Receptores Adrenérgicos beta 2/metabolismo , Transdução de Sinais/efeitos dos fármacos , Células Th17/imunologia , Agonistas de Receptores Adrenérgicos beta 2/farmacologia , Agonistas de Receptores Adrenérgicos beta 2/uso terapêutico , Animais , Proteínas Quinases Dependentes de AMP Cíclico/antagonistas & inibidores , Proteínas Quinases Dependentes de AMP Cíclico/metabolismo , Modelos Animais de Doenças , Regulação para Baixo/efeitos dos fármacos , Masculino , Camundongos , Norepinefrina/farmacologia , Fenótipo , Inibidores de Proteínas Quinases/farmacologia , Células Th17/efeitos dos fármacosRESUMO
Microglia are the main immune cells in the central nervous system. In the present study, the mechanism for acetylcholine (ACh) inhibiting microglial inflammatory response was investigated. Primary culture of microglia was isolated from cerebral cortex of Sprague-Dawley (SD) rats. Lipopolysaccharide (LPS) was used to activate the microglia to induce inflammatory response, and then the microglia were treated with ACh for 24 h. Protein expressions of several inflammatory factors, insulin-like growth factor 1 (IGF-1) and α7 nicotinic acetylcholine receptor (α7nAChR) were detected by Western blot. Release of inflammatory factors and IGF-1 into media was detected by ELISA. After α7nAChR gene silence was achieved by lentivirus-transfection of α7nAChR-shRNA, the change of ACh effect was observed. The results showed that LPS induced microglial activation, up-regulated inducible nitric oxide synthase (iNOS) protein expression, increased the expressions and release of IL-1ß and TNF-α, and decreased the expression and release of the neurotrophic factor, IGF-1. ACh could reverse these effects of LPS. Meanwhile, LPS reduced the protein expression of α7nAChR on the microglial cells, whereas ACh could reverse the effect. Silencing of α7nAChR gene in microglia abolished the ability of ACh to inhibit LPS-induced inflammatory responses. These results suggest that ACh exerts its protection against LPS-induced microglial inflammation via acting on α7nAChR on microglia, which may provide a novel target for the treatment of neuro-inflammatory diseases.
Assuntos
Acetilcolina/farmacologia , Inflamação/tratamento farmacológico , Microglia/efeitos dos fármacos , Fármacos Neuroprotetores/farmacologia , Receptor Nicotínico de Acetilcolina alfa7/metabolismo , Animais , Córtex Cerebral/citologia , Inativação Gênica , Fator de Crescimento Insulin-Like I/metabolismo , Interleucina-1beta/metabolismo , Lipopolissacarídeos , Microglia/citologia , Óxido Nítrico/metabolismo , Óxido Nítrico Sintase Tipo II/metabolismo , Cultura Primária de Células , Ratos , Ratos Sprague-Dawley , Fator de Necrose Tumoral alfa/metabolismoRESUMO
BACKGROUND/AIMS: Regulatory T (Treg) cells have been associated with neuroprotection by inhibiting microglial activation in animal models of Parkinson's disease (PD), a progressive neurodegenerative disease characterized by dopaminergic neuronal loss in the nigrostriatal system. Herein, we show that Treg cells directly protect dopaminergic neurons against 1-methyl-4-phenylpyridinium (MPP+) neurotoxicity via an interaction between the two transmembrane proteins CD47 and signal regulatory protein α (SIRPA). METHODS: Primary ventral mesencephalic (VM) cells or VM neurons were pretreated with Treg cells before MPP+ treatment. Transwell co-culture of Treg cells and VM neurons was used to assess the effects of the Treg cytokines transforming growth factor (TGF)-ß1 and interleukin (IL)-10 on dopaminergic neurons. Live cell imaging system detected a dynamic contact of Treg cells with VM neurons that were stained with CD47 and SIRPA, respectively. Dopaminergic neuronal loss, which was assessed by the number of tyrosine hydroxylase (TH)-immunoreactive cells, was examined after silencing CD47 in Treg cells or silencing SIRPA in VM neurons. RESULTS: Treg cells prevented MPP+-induced dopaminergic neuronal loss and glial inflammatory responses. TGF-ß1 and IL-10 secreted from Treg cells did not significantly prevent MPP+-induced dopaminergic neuronal loss in transwell co-culture of Treg cells and VM neurons. CD47 and SIRPA were expressed by Treg cells and VM neurons, respectively. CD47-labeled Treg cells dynamically contacted with SIRPA-labeled VM neurons. Silencing CD47 gene in Treg cells impaired the ability of Treg cells to protect dopaminergic neurons against MPP+ toxicity. Similarly, SIRPA knockdown in VM neurons reduced the ability of Treg cell neuroprotection. Rac1/Akt signaling pathway in VM neurons was activated by CD47-SIRPA interaction between Treg cells and the neurons. Inhibiting Rac1/Akt signaling in VM neurons compromised Treg cell neuroprotection. CONCLUSION: Treg cells protect dopaminergic neurons against MPP+ neurotoxicity by a cell-to-cell contact mechanism underlying CD47-SIRPA interaction and Rac1/Akt activation.
Assuntos
1-Metil-4-fenilpiridínio/toxicidade , Antígeno CD47/genética , Neurônios Dopaminérgicos/efeitos dos fármacos , Receptores Imunológicos/genética , Linfócitos T Reguladores/efeitos dos fármacos , Animais , Antígeno CD47/imunologia , Comunicação Celular , Morte Celular/efeitos dos fármacos , Técnicas de Cocultura , Cultura em Câmaras de Difusão , Neurônios Dopaminérgicos/citologia , Neurônios Dopaminérgicos/imunologia , Embrião de Mamíferos , Feminino , Expressão Gênica , Interleucina-10/farmacologia , Mesencéfalo/citologia , Mesencéfalo/efeitos dos fármacos , Mesencéfalo/imunologia , Camundongos , Camundongos Endogâmicos C57BL , Neuropeptídeos/genética , Neuropeptídeos/imunologia , Cultura Primária de Células , Proteínas Proto-Oncogênicas c-akt/genética , Proteínas Proto-Oncogênicas c-akt/imunologia , Receptores Imunológicos/imunologia , Transdução de Sinais , Baço/citologia , Baço/efeitos dos fármacos , Baço/imunologia , Linfócitos T Reguladores/citologia , Linfócitos T Reguladores/imunologia , Fator de Crescimento Transformador beta1/farmacologia , Proteínas rac1 de Ligação ao GTP/genética , Proteínas rac1 de Ligação ao GTP/imunologiaRESUMO
Neuroinflammation is associated with pathogenesis of Parkinson's disease (PD), a neurodegenerative disorder characterized by a progressive loss of dopaminergic (DAergic) neurons within the substantia nigra. Transforming growth factor (TGF)-ß1 exerts anti-inflammatory and neuroprotective properties. However, it is unclear if microglia are required for TGF-ß1 neuroprotection in PD. Here we used both shRNA and pharmacologic inhibition to determine the role of microglial TGF-ß receptor (TßR)-I and its downstream signaling pathways in 1-methyl-4-phenylpyridinium (MPP(+))-induced DAergic neuronal toxicity. As expected, MPP(+) reduced the number of tyrosine hydroxylase (TH)-immunoreactive cells in ventral mesencephalic cell cultures. We found that MPP(+) activated microglia as determined by an upregulation in expression of CD11b and inducible nitric oxide synthase (iNOS), an increase in expression and secretion of tumor necrosis factor (TNF)-α and interleukin (IL)-1ß, and a decrease in expression and secretion of the neurotrophic factor, insulin-like growth factor (IGF)-1. Pretreatment with TGF-ß1 significantly inhibited all these changes caused by MPP(+). Expression of microglial TßR-I was upregulated by TGF-ß1. Silencing of the TßR-I gene in microglia abolished both the neuroprotective and anti-inflammatory properties of TGF-ß1. TGF-ß1 increased microglial p38 MAPK and Akt phosphorylation, both of which were blocked by the p38 inhibitor SB203580 and the PI3K inhibitor LY294002, respectively. Pretreatment of microglia with either SB203580 or LY294002 impaired the ability of TGF-ß1 to inhibit MPP(+)-induced DAergic neuronal loss and microglial activation. These findings establish that TGF-ß1 activates TßR-I and its downstream p38 MAPK and PI3K-Akt signaling pathways in microglia to protect against DAergic neuronal loss that characterizes in PD.
Assuntos
1-Metil-4-fenilpiridínio/farmacologia , Neurônios Dopaminérgicos/efeitos dos fármacos , Neurônios Dopaminérgicos/metabolismo , Microglia/metabolismo , Transtornos Parkinsonianos/metabolismo , Proteínas Serina-Treonina Quinases/metabolismo , Receptores de Fatores de Crescimento Transformadores beta/metabolismo , Transdução de Sinais/efeitos dos fármacos , Fator de Crescimento Transformador beta1/farmacologia , Animais , Astrócitos/metabolismo , Células Cultivadas , Córtex Cerebral/efeitos dos fármacos , Córtex Cerebral/metabolismo , Mediadores da Inflamação/metabolismo , Sistema de Sinalização das MAP Quinases/efeitos dos fármacos , Mesencéfalo/efeitos dos fármacos , Mesencéfalo/metabolismo , Camundongos , Camundongos Endogâmicos C57BL , Fosfatidilinositol 3-Quinases/metabolismo , Proteínas Serina-Treonina Quinases/genética , Proteínas Proto-Oncogênicas c-akt/metabolismo , Receptor do Fator de Crescimento Transformador beta Tipo I , Receptores de Fatores de Crescimento Transformadores beta/genéticaRESUMO
OBJECTIVES: We have previously reported that dopamine D2-like receptors including D2, D3 and D4 receptors are more important in mediating modulation of T cells than dopamine D1-like receptors (D1 and D5 receptors). Here we aimed to clarify the role of D2-like receptors in regulation of differentiation and function of T lymphocyte subsets, including helper T (Th)1, Th2, Th17 and regulatory T (Treg) cells. METHODS: Lymphocytes, separated from the mesenteric lymph nodes of mice, were stimulated with concanavalin A (Con A) and treated with the D2-like receptor agonist quinpirole or the antagonist haloperidol. Expression of lymphocyte cytokines and transcription factors and dopamine D2, D3 and D4 receptors were measured by real-time quantitative polymerase chain reaction and Western blot assay. Meanwhile, cAMP and phosphorylated cAMP-response element-binding (CREB) levels in the lymphocytes were examined by enzyme-linked immunosorbent assay and Western blot assay, respectively. RESULTS: Activation of D2-like receptors with the agonist quinpirole upregulated the expression of Th2- and Treg-specific transcription factors and cytokines in Con A-activated lymphocytes, but downregulated the expression of Th1- and Th17-specific transcription factors and cytokines. Simultaneously, quinpirole increased dopamine D3 and D4 receptor expression, but did not alter D2 receptor expression. However, quinpirole reduced both cAMP and phosphorylated CREB levels in Con A-activated lymphocytes. All these quinpirole effects were blocked by haloperidol, an antagonist of D2-like receptors. CONCLUSIONS: D2-like receptors, principally dopamine D3 and D4 receptors, promote differentiation and function of T lymphocytes towards anti-inflammatory T cell subsets by a negative link to cAMP-CREB pathway.
Assuntos
Receptores Dopaminérgicos/metabolismo , Transdução de Sinais/efeitos dos fármacos , Linfócitos T/metabolismo , Animais , AMP Cíclico/metabolismo , Modulador de Elemento de Resposta do AMP Cíclico/metabolismo , Agonistas de Dopamina/farmacologia , Antagonistas de Dopamina/farmacologia , Ensaio de Imunoadsorção Enzimática , Camundongos Endogâmicos ICR , Fosforilação , Quimpirol/farmacologia , Receptores Dopaminérgicos/efeitos dos fármacos , Linfócitos T/efeitos dos fármacosRESUMO
Curing patients with acute myeloid leukemia (AML) remains a therapeutic challenge. The polycomb complex protein B-cell-specific Moloney murine leukemia virus integration site 1 (BMI-1) is required for the self-renewal and maintenance of leukemia stem cells. We investigated the prognostic significance of BMI-1 in AML and the effects of a novel small molecule selective inhibitor of BMI-1, PTC-209. BMI-1 protein expression was determined in 511 newly diagnosed AML patients together with 207 other proteins using reverse-phase protein array technology. Patients with unfavorable cytogenetics according to Southwest Oncology Group criteria had higher levels of BMI-1 compared to those with favorable (P = 0.0006) or intermediate cytogenetics (P = 0.0061), and patients with higher levels of BMI-1 had worse overall survival (55.3 weeks vs. 42.8 weeks, P = 0.046). Treatment with PTC-209 reduced protein level of BMI-1 and its downstream target mono-ubiquitinated histone H2A and triggered several molecular events consistent with the induction of apoptosis, this is, loss of mitochondrial membrane potential, caspase-3 cleavage, BAX activation, and phosphatidylserine externalization. PTC-209 induced apoptosis in patient-derived CD34(+)CD38(low/-) AML cells and, less prominently, in CD34(-) differentiated AML cells. BMI-1 reduction by PTC-209 directly correlated with apoptosis induction in CD34(+) primary AML cells (r = 0.71, P = 0.022). However, basal BMI-1 expression was not a determinant of AML sensitivity. BMI-1 inhibition, which targets a primitive AML cell population, might offer a novel therapeutic strategy for AML.
Assuntos
Antineoplásicos/farmacologia , Compostos Heterocíclicos com 2 Anéis/farmacologia , Leucemia Mieloide Aguda/patologia , Complexo Repressor Polycomb 1/biossíntese , Tiazóis/farmacologia , Apoptose/efeitos dos fármacos , Western Blotting , Ciclo Celular/efeitos dos fármacos , Linhagem Celular Tumoral , Citometria de Fluxo , Humanos , Estimativa de Kaplan-Meier , Leucemia Mieloide Aguda/mortalidade , Prognóstico , RNA Interferente Pequeno , Reação em Cadeia da Polimerase em Tempo Real , TransfecçãoRESUMO
Chromosomal region maintenance 1 (CRM1) is a nuclear export receptor recognizing proteins bearing a leucine-rich nuclear export signal. CRM1 is involved in nuclear export of tumor suppressors such as p53. We investigated the prognostic significance of CRM1 in acute myeloid leukemia (AML) and effects of a novel small-molecule selective inhibitor of CRM1. CRM1 protein expression was determined in 511 newly diagnosed AML patients and was correlated with mouse double minute 2 (MDM2) and p53 levels. High CRM1 expression was associated with short survival of patients and remained an adverse prognostic factor in multivariate analysis. CRM1 inhibitor KPT-185 induced mainly full-length p53 and apoptosis in a p53-dependent manner, whereas inhibition of proliferation was p53 independent. Patient samples with p53 mutations showed low sensitivity to KPT-185. Nuclear retention of p53 induced by CRM1 inhibition synergized with increased levels of p53 induced by MDM2 inhibition in apoptosis induction. KPT-185 and Nutlin-3a, alone and in combination, induced synergistic apoptosis in patient-derived CD34(+)/CD38(-) AML, but not in normal progenitor cells. Data suggest that CRM1 exerts an antiapoptotic function and is highly prognostic in AML. We propose a novel combinatorial approach for the therapy of AML, aimed at maximal activation of p53-mediated apoptosis by concomitant MDM2 and CRM1 inhibition.
Assuntos
Acrilatos/uso terapêutico , Antineoplásicos/uso terapêutico , Biomarcadores Tumorais , Carioferinas/antagonistas & inibidores , Carioferinas/fisiologia , Leucemia Mieloide Aguda/diagnóstico , Leucemia Mieloide Aguda/tratamento farmacológico , Receptores Citoplasmáticos e Nucleares/antagonistas & inibidores , Receptores Citoplasmáticos e Nucleares/fisiologia , Triazóis/uso terapêutico , Biomarcadores Tumorais/genética , Biomarcadores Tumorais/fisiologia , Células Cultivadas , Avaliação Pré-Clínica de Medicamentos , Resistencia a Medicamentos Antineoplásicos/genética , Feminino , Células HL-60 , Humanos , Carioferinas/genética , Leucemia Mieloide Aguda/genética , Masculino , Terapia de Alvo Molecular , Prognóstico , Receptores Citoplasmáticos e Nucleares/genética , Proteína Supressora de Tumor p53/genética , Proteína Supressora de Tumor p53/metabolismo , Proteína Supressora de Tumor p53/fisiologia , Células U937 , Proteína Exportina 1RESUMO
OBJECTIVE: Recently, we have reported that lymphocyte-derived endogenous catecholamines (CAs) facilitate a shift in the T helper (Th)1/Th2 balance towards Th2. The purpose of this study was to explore the involvement of adrenoreceptors (ARs) in Th differentiation and function modulation by lymphocyte-derived CAs. METHODS: Lymphocytes were separated from the mesenteric lymph nodes of mice, stimulated with concanavalin A (Con A) and treated with pargyline, an inhibitor of CA degradation. RESULTS: Pargyline downregulated the expression of Th1-relative factors, T-bet, interferon (IFN)-γ and interleukin (IL)-2, but upregulated the expression of Th2-relative factors, GATA-3, IL-4 and IL-10. Pargyline reduced the percentage of IFN-γ-producing CD4+ cells and the CD4+IFN-γ+/CD4+IL-4+ cell ratio, although it did not alter the proportion of IL-4-producing CD4+ cells. In addition, the percentage of CD4+CD26+ T cells and the CD4+CD26+/CD4+CD30+ cell ratio were also reduced in the pargyline-treated group. Furthermore, Con A-activated T cells treated with pargyline produced a lower level of IFN-γ and a higher level of IL-4 than the control group. All these effects were blocked by the α1-AR antagonist corynanthine or the ß2-AR antagonist ICI 118551, but not by the α2-AR antagonist yohimbine or ß1-AR antagonist atenolol. CONCLUSIONS: These results imply that lymphocyte-derived CAs promote polarization of differentiation and function towards Th2 cells and that this effect is mediated by α1-AR and ß2-AR.
Assuntos
Catecolaminas/metabolismo , Diferenciação Celular/fisiologia , Linfócitos/metabolismo , Receptores Adrenérgicos alfa 1/metabolismo , Receptores Adrenérgicos beta 2/metabolismo , Linfócitos T Auxiliares-Indutores/fisiologia , Adrenérgicos/farmacologia , Animais , Diferenciação Celular/efeitos dos fármacos , Citocinas/genética , Citocinas/metabolismo , Citometria de Fluxo , Fator de Transcrição GATA3/genética , Fator de Transcrição GATA3/metabolismo , Linfócitos/efeitos dos fármacos , Camundongos , Camundongos Endogâmicos ICR , Inibidores da Monoaminoxidase/farmacologia , Pargilina/farmacologia , RNA Mensageiro/metabolismo , Linfócitos T Auxiliares-Indutores/efeitos dos fármacosRESUMO
OBJECTIVE: We previously showed that the cerebellum modulates the immune system. Here we determined whether cerebellar ataxia alters immunological function to further demonstrate an involvement of the cerebellum in immune modulation. METHODS: Neurotoxin 3-acetylpyridine (3-AP) was intraperitoneally injected in rats to induce cerebellar ataxia. Behavior and motor coordination were tested on day 7 following 3-AP injection. Nissl staining and high-performance liquid chromatography (HPLC) were used to determine neuronal loss and neurotransmitter contents, respectively, in all the three cerebellar nuclei, fastigial nucleus (FN), interposed nucleus (IN) and dentate nucleus (DN). T and B lymphocyte differentiation and function were measured by flow cytometry, Western blot and ELISA. RESULTS: 3-AP induced motor discoordination and locomotor reduction. In all the three cerebellar nuclei, FN, IN and DN, there was a neuronal loss and a decrease in contents of glutamate and GABA (but not glycine) after 3-AP injection. Importantly, CD4+ T cells, but not CD8+ T cells, were increased by the 3-AP treatment. Moreover, interferon (IFN)-γ-producing cells and interleukin (IL)-17-producing cells were decreased in cerebellar ataxia rats, but IL-4-producing cells and CD25-expressing cells were increased. Expression of the T helper (Th)1- and Th17-related cytokines, IFN-γ, IL-2, IL-17 and IL-22, was downregulated in CD4+ cells in cerebellar ataxia rats, while expression of the Th2 and regulatory T (Treg)-related cytokines, IL-4, IL-5, IL-10 and transforming growth factor (TGF)-ß, was upregulated. Furthermore, B lymphocyte number and anti-bovine serum albumin (BSA) IgM and IgG antibody levels were elevated in cerebellar ataxia. CONCLUSION: Cerebellar ataxia alters cellular and humoral immunity.
Assuntos
Ataxia Cerebelar/imunologia , Ataxia Cerebelar/metabolismo , Imunidade Celular/imunologia , Imunidade Humoral/imunologia , Animais , Ataxia Cerebelar/induzido quimicamente , Modelos Animais de Doenças , Masculino , Ratos , Ratos Sprague-DawleyRESUMO
Interleukin (IL)-10, an anti-inflammatory cytokine, is expressed in the brain and can inhibit microglial activation. Herein, we utilized lipopolysaccharide (LPS)-induced inflammatory Parkinson's disease (PD) cell model to determine whether microglia and astrocytes are necessary targets for IL-10 neuroprotection. Primary ventral mesencephalic (VM) cultures with different composition of neurons, microglia and astrocytes were prepared. The cells were exposed to IL-10 (15, 50 or 150 ng/mL) 1 h prior to LPS (50 ng/mL) treatment. LPS induced dopaminergic and non-dopaminergic neuronal loss in VM cultures, VM neuron-enriched cultures, and neuron-microglia co-cultures, but not in neuron-astrocyte co-cultures. IL-10 reduced LPS-induced neuronal loss particularly in single VM neuron cultures. Pro-inflammatory mediators (TNF-α, IL-1ß, inducible nitric oxide synthase and cyclooxygenase-2) were upregulated in both neuron-microglia and neuron-astrocyte co-cultures by LPS. In contrast, neurotrophic factors (brain-derived neurotrophic factor, insulin-like growth factor-1 or glial cell-derived neurotrophic factor) were downregulated in neuron-microglia co-cultures, but upregulated in neuron-astrocyte co-cultures by LPS. IL-10 reduced both the increase in production of the pro-inflammatory mediators and the decrease in production of the neurotrophic factors induced by LPS. These results suggest that astrocytes can balance LPS neurotoxicity by releasing more neurotrophic factors and that IL-10 exerts neuroprotective property by an extensive action including direct on neurons and indirect via inhibiting microglial activation.
Assuntos
Inflamação/induzido quimicamente , Interleucina-10/farmacologia , Lipopolissacarídeos/efeitos adversos , Mesencéfalo/metabolismo , Síndromes Neurotóxicas/etiologia , Doença de Parkinson/etiologia , Animais , Anti-Inflamatórios/farmacologia , Astrócitos/imunologia , Astrócitos/metabolismo , Técnicas de Cocultura , Inflamação/imunologia , Mesencéfalo/imunologia , Microglia/imunologia , Microglia/metabolismo , Neurônios/imunologia , Neurônios/metabolismo , Síndromes Neurotóxicas/imunologia , Síndromes Neurotóxicas/metabolismo , Doença de Parkinson/imunologia , Doença de Parkinson/metabolismo , Ratos , Ratos Sprague-DawleyRESUMO
Our recent work has shown that the cerebellar interposed nucleus (IN) contains glutamatergic neurons that send axons directly to the hypothalamus. In the present study, we aimed to demonstrate modulation of cellular and humoral immunity by glutamatergic neurons in the cerebellar IN by means of gene interventions of glutaminase (GLS), an enzyme for glutamate synthesis, and to reveal pathways transmitting the immunomodulation. Injection of GLS-shRNA lentiviral vector into bilateral cerebellar IN downregulated GLS expression in the IN. The silencing of GLS gene in the cerebellar IN decreased interleukin (IL)-2 and interferon (IFN)-γ production, B-cell number, and IgM antibody level in response to antigen bovine serum albumin (BSA). On the contrary, injection of GLS lentiviral vector into bilateral cerebellar IN upregulated GLS expression in the IN. The GLS gene overexpression in the IN caused opposite immune effects to the GLS gene knockdown. Simultaneously, the GLS gene silencing in the cerebellar IN reduced and the GLS overexpression elevated glutamate content in the hypothalamus, but they both did not affect glycine and GABA contents in the hypothalamus. In addition, the immune changes caused by the GLS gene interventions in the IN were accompanied by alteration in norepinephrine content in the spleen and mesenteric lymph nodes but not by changes in adrenocortical and thyroid hormone levels in serum. These findings indicate that glutamatergic neurons in the cerebellar IN regulate cellular and humoral immune responses and suggest that such immunoregulation may be conveyed by cerebellar IN-hypothalamic glutamatergic projections and sympathetic nerves that innervate lymphoid tissues.
Assuntos
Núcleos Cerebelares/imunologia , Ácido Glutâmico/metabolismo , Hipotálamo/imunologia , Neurônios/imunologia , Animais , Núcleos Cerebelares/metabolismo , Citocinas/análise , Glutaminase/genética , Hipotálamo/metabolismo , Camundongos Transgênicos , Vias Neurais , Ratos , Ratos Sprague-Dawley , Sistema Nervoso Simpático/imunologia , Sistema Nervoso Simpático/metabolismoRESUMO
Laggeric acid (1), a novel 4,5-seco-eudesmane sesquiterpene acid, along with 19 known compounds, was isolated from the MeOH extract of the whole plant of Laggera crispata. The structure of 1 was elucidated to be (4S,5S*,7R*,10R*)-4,5-seco-4,5-dihydroxy-11(13)-eudesmen-12-oic acid on the basis of spectroscopic analysis and a convenient Mosher's method.
Assuntos
Medicamentos de Ervas Chinesas/isolamento & purificação , Sesquiterpenos de Eudesmano/isolamento & purificação , Asteraceae/química , Medicamentos de Ervas Chinesas/química , Estrutura Molecular , Ressonância Magnética Nuclear Biomolecular , Sesquiterpenos , Sesquiterpenos de Eudesmano/química , EstereoisomerismoRESUMO
Transforming growth factor (TGF)-ß1, a cytokine that can be expressed in the brain, is a key regulator of the brain's responses to injury and inflammation. Alzheimer's disease (AD), the most common neurodegenerative disorder, involves inflammatory processes in the brain in addition to the hallmarks, amyloid-ß (Aß) plaques and neurofibrillary tangles. Recently, we have shown that T-helper (Th) 17 cells, a subpopulation of CD4+ T-cells with high proinflammation, also participate in the brain inflammatory process of AD. However, it is poorly known whether TGF-ß1 ameliorates the lymphocyte-mediated neuroinflammation and, thereby, alleviates neurodegeneration in AD. Herein, we administered TGF-ß1 via the intracerebroventricle (ICV) in AD model rats, by Aß1-42 injection in both sides of the hippocampus, to show the neuroprotection of TGF-ß1. The TGF-ß1 administration after the Aß1-42 injection ameliorated cognitive deficit and neuronal loss and apoptosis, reduced amyloid precursor protein (APP) expression, elevated protein phosphatase (PP)2A expression, attenuated glial activation and alleviated the imbalance of the pro-inflammatory/anti-inflammatory responses of T-lymphocytes, compared to the Aß1-42 injection alone. These findings demonstrate that TGF-ß1 provides protection against AD neurodegeneration and suggest that the TGF-ß1 neuroprotection is implemented by the alleviation of glial and T-cell-mediated neuroinflammation.
Assuntos
Peptídeos beta-Amiloides/toxicidade , Encéfalo/patologia , Inflamação/tratamento farmacológico , Inflamação/patologia , Degeneração Neural/tratamento farmacológico , Degeneração Neural/patologia , Fármacos Neuroprotetores/uso terapêutico , Fragmentos de Peptídeos/toxicidade , Fator de Crescimento Transformador beta1/uso terapêutico , Animais , Encéfalo/efeitos dos fármacos , Encéfalo/fisiopatologia , Cognição/efeitos dos fármacos , Citocinas/sangue , Citocinas/líquido cefalorraquidiano , Regulação para Baixo/efeitos dos fármacos , Hipocampo/efeitos dos fármacos , Hipocampo/patologia , Hipocampo/fisiopatologia , Hipotálamo/efeitos dos fármacos , Hipotálamo/patologia , Hipotálamo/fisiopatologia , Inflamação/sangue , Inflamação/complicações , Mediadores da Inflamação/metabolismo , Degeneração Neural/sangue , Degeneração Neural/fisiopatologia , Neurônios/efeitos dos fármacos , Neurônios/patologia , Fármacos Neuroprotetores/farmacologia , Ratos Sprague-Dawley , Linfócitos T/efeitos dos fármacos , Linfócitos T/metabolismo , Fator de Crescimento Transformador beta1/farmacologia , Regulação para Cima/efeitos dos fármacosRESUMO
Regulators of apoptosis in acute myeloid leukemia (AML) have been extensively studied and are considered excellent therapeutic targets. Apoptosis repressor with caspase recruitment domain (ARC), an antiapoptotic protein originally found to be involved in apoptosis of cardiac cells, was recently demonstrated to be overexpressed in several solid tumors. To assess its importance in AML, we profiled ARC expression in 511 newly diagnosed AML patients using a validated robust reverse-phase protein array and correlated ARC levels with clinical outcomes. ARC was variably expressed in samples from patients with AML. ARC level was not associated with cytogenetic groups or with FLT-3 mutation status. However, patients with low or medium ARC protein levels had significantly better outcomes than those with high ARC levels: longer overall survival (median, 53.9 or 61.6 vs 38.9 weeks, P = .0015) and longer remission duration (median, 97.6 or 44.7 vs 31.1 weeks, P = .0007). Multivariate analysis indicated that ARC was a statistically significant independent predictor of survival in AML (P = .00013). Inhibition of ARC promoted apoptosis and sensitized cytosine arabinoside-induced apoptosis in OCI-AML3 cells. These results suggest that ARC expression levels are highly prognostic in AML and that ARC is a potential therapeutic target in AML.
Assuntos
Proteínas Reguladoras de Apoptose/metabolismo , Leucemia Mieloide/metabolismo , Proteínas Musculares/metabolismo , Doença Aguda , Adolescente , Adulto , Idoso , Idoso de 80 Anos ou mais , Apoptose/efeitos dos fármacos , Proteínas Reguladoras de Apoptose/genética , Western Blotting , Linhagem Celular Tumoral , Sobrevivência Celular/efeitos dos fármacos , Citarabina/farmacologia , Feminino , Humanos , Leucemia Mieloide/diagnóstico , Leucemia Mieloide/genética , Masculino , Pessoa de Meia-Idade , Análise Multivariada , Proteínas Musculares/genética , Mutação , Oligonucleotídeos Antissenso/genética , Prognóstico , Análise de Sobrevida , Adulto Jovem , Tirosina Quinase 3 Semelhante a fms/genéticaRESUMO
Friend leukemia virus integration 1 (FLI1), an Ets transcription factor family member, is linked to acute myelogenous leukemia (AML) by chromosomal events at the FLI1 locus, but the biologic impact of FLI1 expression on AML is unknown. FLI1 protein expression was measured in 511 newly diagnosed AML patients. Expression was similar in peripheral blood (PB) and BM and higher at diagnosis than at relapse (P = .02). Compared with normal CD34(+) cells, expression in AML was above or below normal in 32% and 5% of patients, respectively. Levels were negatively correlated with an antecedent hematologic disorder (P = .002) but not with age or cytogenetics. Mutated NPM1 (P = .0007) or FLT3-ITD (P < .02) had higher expression. FLI1 levels were negatively correlated with 10 of 195 proteins associated with proliferation and stromal interaction, and positively correlated (R > 0.3) with 19 others. The FLI1 level was not predictive of remission attainment, but patients with low or high FLI1 expression had shorter remission duration (22.6 and 40.3 vs 51.1 weeks, respectively; P = .01) and overall survival (45.2 and 35.4 vs 59.4 weeks, respectively; P = .03). High FLI1 levels were adverse in univariate and multivariate analysis. FLI1 expression is frequently abnormal and prognostically adverse in AML. FLI1 and/or its response genes may be therapeutically targetable to interfere with AML cell biology.
Assuntos
Regulação Leucêmica da Expressão Gênica/genética , Leucemia Mieloide Aguda/genética , Leucemia Mieloide Aguda/mortalidade , Proteína Proto-Oncogênica c-fli-1/genética , Adolescente , Adulto , Idoso , Idoso de 80 Anos ou mais , Criança , Pré-Escolar , Feminino , Humanos , Estimativa de Kaplan-Meier , Masculino , Pessoa de Meia-Idade , Proteínas Nucleares/genética , Nucleofosmina , Prognóstico , Proteômica , Fatores de Risco , Adulto Jovem , Tirosina Quinase 3 Semelhante a fms/genéticaRESUMO
We have previously shown that interleukin-6 (IL-6) has neuroprotective effect against N-methyl-D-aspartate (NMDA)-induced excitotoxicity. The current study aimed to reveal signal transduction pathways involved in the IL-6 neuroprotection. Cerebellar granule neurons (CGNs) from postnatal 8-day infant rats were exposed to IL-6 (120 ng/ml) for 8 days and stimulated with NMDA (100 µM) for 15 or 30 min. Dynamic intracellular Ca(2+) fluorescence intensity, cytosolic Ca(2+)-dependent phospholipase A2 (cPLA2) expression, and apoptosis and necrosis in cultured CGNs were measured by laser scanning confocal microscope, real-time PCR and Western blot, and annexin V-FITC/propidium iodide staining, respectively. NMDA stimulation of neurons evoked an intracellular Ca(2+) overload, an upregulated expression of cPLA2, and an increase in cell death. Chronic IL-6 exposure prevented the NMDA-evoked neuronal Ca(2+) overload, cPLA2 expression upregulation, and apoptosis and necrosis. Anti-gp130 monoclonal antibody (mAb), a blocker of gp130 that is a 130-kDa signal-transducing ß-subunit of IL-6 receptor complex, blocked these effects of IL-6 preventing NMDA neurotoxicity. AG490, PD98059, or LY294002, inhibitors specific for the intracellular signals, JAK, MAPK, and PI3K, respectively, partially blocked these IL-6 neuroprotective effects. Phosphorylation levels of STAT3, ERK1/2, and AKT, the downstream proteins for these enzymes of JAK, MAPK, and PI3K, respectively, were elevated by IL-6 pretreatment. The enhanced activation of STAT3, ERK1/2, and AKT by IL-6 was abolished by AG490, PD98059, and LY294002, respectively. Anti-gp130 mAb attenuated the activation of all the three detected signaling molecules. The present findings suggest that IL-6 neuroprotection is jointly mediated by the cellular signal transduction pathways, gp130-JAK-STAT3, gp130-MAPK-ERK, and gp130-PI3K-AKT.