RESUMO
Long-term epigenetic reprogramming of innate immune cells in response to microbes, also termed "trained immunity," causes prolonged altered cellular functionality to protect from secondary infections. Here, we investigated whether sterile triggers of inflammation induce trained immunity and thereby influence innate immune responses. Western diet (WD) feeding of Ldlr-/- mice induced systemic inflammation, which was undetectable in serum soon after mice were shifted back to a chow diet (CD). In contrast, myeloid cell responses toward innate stimuli remained broadly augmented. WD-induced transcriptomic and epigenomic reprogramming of myeloid progenitor cells led to increased proliferation and enhanced innate immune responses. Quantitative trait locus (QTL) analysis in human monocytes trained with oxidized low-density lipoprotein (oxLDL) and stimulated with lipopolysaccharide (LPS) suggested inflammasome-mediated trained immunity. Consistently, Nlrp3-/-/Ldlr-/- mice lacked WD-induced systemic inflammation, myeloid progenitor proliferation, and reprogramming. Hence, NLRP3 mediates trained immunity following WD and could thereby mediate the potentially deleterious effects of trained immunity in inflammatory diseases.
Assuntos
Reprogramação Celular , Dieta Ocidental , Epigênese Genética , Imunidade Inata , Memória Imunológica , Proteína 3 que Contém Domínio de Pirina da Família NLR/genética , Adulto , Idoso , Animais , Células Cultivadas , Feminino , Humanos , Lipoproteínas LDL/metabolismo , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Pessoa de Meia-Idade , Células Mieloides/imunologia , Proteína 3 que Contém Domínio de Pirina da Família NLR/metabolismo , Locos de Características Quantitativas , Receptores de LDL/genéticaRESUMO
High-density lipoprotein (HDL) mediates reverse cholesterol transport and is known to be protective against atherosclerosis. In addition, HDL has potent anti-inflammatory properties that may be critical for protection against other inflammatory diseases. The molecular mechanisms of how HDL can modulate inflammation, particularly in immune cells such as macrophages, remain poorly understood. Here we identify the transcriptional regulator ATF3, as an HDL-inducible target gene in macrophages that downregulates the expression of Toll-like receptor (TLR)-induced proinflammatory cytokines. The protective effects of HDL against TLR-induced inflammation were fully dependent on ATF3 in vitro and in vivo. Our findings may explain the broad anti-inflammatory and metabolic actions of HDL and provide the basis for predicting the success of new HDL-based therapies.
Assuntos
Fator 3 Ativador da Transcrição/metabolismo , Anti-Inflamatórios não Esteroides/uso terapêutico , Aterosclerose/terapia , Colesterol/metabolismo , Inflamação/terapia , Lipoproteínas HDL/uso terapêutico , Macrófagos/efeitos dos fármacos , Fator 3 Ativador da Transcrição/genética , Animais , Anti-Inflamatórios não Esteroides/farmacologia , Células Cultivadas , Imunoprecipitação da Cromatina , Citocinas/metabolismo , Feminino , Perfilação da Expressão Gênica , Sequenciamento de Nucleotídeos em Larga Escala , Humanos , Lipoproteínas HDL/farmacologia , Ativação de Macrófagos/efeitos dos fármacos , Macrófagos/imunologia , Camundongos , Camundongos Endogâmicos C3H , Camundongos Endogâmicos C57BL , Biologia de Sistemas , Receptor 4 Toll-Like/genética , Receptor 4 Toll-Like/imunologiaRESUMO
Carrying premature termination codons in 1 allele of the ABCA7 gene is associated with an increased risk for Alzheimer's disease (AD). While the primary function of ABCA7 is to regulate the transport of phospholipids and cholesterol, ABCA7 is also involved in maintaining homeostasis of the immune system. Since inflammatory pathways causatively or consequently participate in AD pathogenesis, we studied the effects of Abca7 haplodeficiency in mice on brain immune responses under acute and chronic conditions. When acute inflammation was induced through peripheral lipopolysaccharide injection in control or heterozygous Abca7 knockout mice, partial ABCA7 deficiency diminished proinflammatory responses by impairing CD14 expression in the brain. On breeding to AppNL-G-F knockin mice, we observed increased amyloid-ß (Aß) accumulation and abnormal endosomal morphology in microglia. Taken together, our results demonstrate that ABCA7 loss of function may contribute to AD pathogenesis by altering proper microglial responses to acute inflammatory challenges and during the development of amyloid pathology, providing insight into disease mechanisms and possible treatment strategies.
Assuntos
Transportadores de Cassetes de Ligação de ATP/genética , Encéfalo/imunologia , Haploinsuficiência , Microglia/imunologia , Doença de Alzheimer/metabolismo , Peptídeos beta-Amiloides/metabolismo , Animais , Perfilação da Expressão Gênica , Imunidade Inata/genética , Camundongos , Camundongos Transgênicos , TranscriptomaRESUMO
Weddell and elephant seals are deep-diving mammals, which rely on lung collapse to limit nitrogen absorption and prevent decompression injury. Repeated collapse and re-expansion exposes the lungs to multiple stressors, including ischemia-reperfusion, alveolar shear stress and inflammation. There is no evidence, however, that diving damages pulmonary function in these species. To investigate potential protective strategies in deep-diving seals, we examined the inflammatory response of seal whole blood exposed to lipopolysaccharide (LPS), a potent endotoxin. Interleukin-6 (IL6) cytokine production elicited by LPS exposure was 50 to 500 times lower in blood of healthy northern elephant seals and Weddell seals compared with that of healthy human blood. In contrast to the â¼6× increased production of IL6 protein from LPS-exposed Weddell seal whole blood, isolated Weddell seal peripheral blood mononuclear cells, under standard cell culture conditions using medium supplemented with fetal bovine serum (FBS), produced a robust LPS response (â¼300×). Induction of Il6 mRNA expression as well as production of IL6, IL8, IL10, KC-like and TNFα were reduced by substituting FBS with an equivalent amount of autologous seal serum. Weddell seal serum also attenuated the inflammatory response of RAW 267.4 mouse macrophage cells exposed to LPS. Cortisol level and the addition of serum lipids did not impact the cytokine response in cultured cells. These data suggest that seal serum possesses anti-inflammatory properties, which may protect deep divers from naturally occurring inflammatory challenges such as dive-induced hypoxia-reoxygenation and lung collapse.
Assuntos
Anti-Inflamatórios/imunologia , Citocinas/metabolismo , Imunidade Inata , Lipopolissacarídeos/farmacologia , Focas Verdadeiras/imunologia , Soro/imunologia , Animais , Anti-Inflamatórios/sangue , Mergulho/fisiologia , Feminino , Leucócitos/imunologia , Masculino , Focas Verdadeiras/sangue , Especificidade da EspécieRESUMO
In Alzheimer's disease (AD), the accumulation and deposition of amyloid-ß (Aß) peptides in the brain is a central event. Aß is cleaved from amyloid precursor protein (APP) by ß-secretase and γ-secretase mainly in neurons. Although mutations inAPP,PS1, orPS2cause early-onset familial AD,ABCA7encoding ATP-binding cassette transporter A7 is one of the susceptibility genes for late-onset AD (LOAD), in which itsloss-of-functionvariants increase the disease risk. ABCA7 is homologous to a major lipid transporter ABCA1 and is highly expressed in neurons and microglia in the brain. Here, we show that ABCA7 deficiency altered brain lipid profile and impaired memory in ABCA7 knock-out (Abca7(-/-)) mice. When bred to amyloid model APP/PS1 mice, plaque burden was exacerbated by ABCA7 deficit.In vivomicrodialysis studies indicated that the clearance rate of Aß was unaltered. Interestingly, ABCA7 deletion facilitated the processing of APP to Aß by increasing the levels of ß-site APP cleaving enzyme 1 (BACE1) and sterol regulatory element-binding protein 2 (SREBP2) in primary neurons and mouse brains. Knock-down of ABCA7 expression in neurons caused endoplasmic reticulum stress highlighted by increased level of protein kinase R-like endoplasmic reticulum kinase (PERK) and increased phosphorylation of eukaryotic initiation factor 2α (eIF2α). In the brains of APP/PS1;Abca7(-/-)mice, the level of phosphorylated extracellular regulated kinase (ERK) was also significantly elevated. Together, our results reveal novel pathways underlying the association of ABCA7 dysfunction and LOAD pathogenesis. SIGNIFICANCE STATEMENT: Gene variants inABCA7encoding ATP-binding cassette transporter A7 are associated with the increased risk for late-onset Alzheimer's disease (AD). Importantly, we found the altered brain lipid profile and impaired memory in ABCA7 knock-out mice. The accumulation of amyloid-ß (Aß) peptides cleaved from amyloid precursor protein (APP) in the brain is a key event in AD pathogenesis and we also found that ABCA7 deficit exacerbated brain Aß deposition in amyloid AD model APP/PS1 mice. Mechanistically, we found that ABCA7 deletion facilitated the processing of APP and Aß production by increasing the levels of ß-secretase 1 (BACE1) in primary neurons and mouse brains without affecting the Aß clearance rate in APP/PS1 mice. Our study demonstrates a novel mechanism underlying how dysfunctions of ABCA7 contribute to the risk for AD.
Assuntos
Transportadores de Cassetes de Ligação de ATP/deficiência , Doença de Alzheimer , Peptídeos beta-Amiloides/metabolismo , Encéfalo/metabolismo , Regulação da Expressão Gênica/genética , Transportadores de Cassetes de Ligação de ATP/genética , Transportadores de Cassetes de Ligação de ATP/metabolismo , Doença de Alzheimer/genética , Doença de Alzheimer/metabolismo , Doença de Alzheimer/patologia , Precursor de Proteína beta-Amiloide/genética , Animais , Encéfalo/patologia , Modelos Animais de Doenças , Fator de Iniciação 2 em Eucariotos/metabolismo , Feminino , Humanos , Metabolismo dos Lipídeos/genética , Masculino , Transtornos da Memória/genética , Camundongos , Camundongos Transgênicos , Mutação/genética , Presenilina-1/genética , Transdução de Sinais/genéticaRESUMO
Macrophage ABCA1 effluxes lipid and has anti-inflammatory activity. The syntrophins, which are cytoplasmic PDZ protein scaffolding factors, can bind ABCA1 and modulate its activity. However, many of the data assessing the function of the ABCA1-syntrophin interaction are based on overexpression in nonmacrophage cells. To assess endogenous complex function in macrophages, we derived immortalized macrophages from Abca1(+/+) and Abca1(-/-) mice and show their phenotype recapitulates primary macrophages. Abca1(+/+) lines express the CD11B and F4/80 macrophage markers and markedly upregulate cholesterol efflux in response to LXR nuclear hormone agonists. In contrast, immortalized Abca1(-/-) macrophages show no efflux to apoA-I. In response to LPS, Abca1(-/-) macrophages display pro-inflammatory changes, including an increased level of expression of cell surface CD14, and 11-26-fold higher levels of IL-6 and IL-12 mRNA. Given recapitulation of phenotype, we show with these lines that the ABCA1-syntrophin protein complex is upregulated by LXR agonists and can bind apoA-I. Moreover, in immortalized macrophages, combined α1/ß2-syntrophin loss modulated ABCA1 cell surface levels and induced pro-inflammatory gene expression. However, loss of all three syntrophin isoforms known to bind ABCA1 did not impair lipid efflux in immortalized or primary macrophages. Thus, the ABCA1-syntrophin protein complex is not essential for ABCA1 macrophage lipid efflux but does directly interact with apoA-I and can modulate the pool of cell surface ABCA1 stabilized by apoA-I.
Assuntos
Transportador 1 de Cassete de Ligação de ATP/metabolismo , Apolipoproteína A-I/metabolismo , Proteínas Associadas à Distrofina/metabolismo , Macrófagos/metabolismo , Receptores Nucleares Órfãos/agonistas , Transportador 1 de Cassete de Ligação de ATP/deficiência , Transportador 1 de Cassete de Ligação de ATP/genética , Animais , Transporte Biológico Ativo , Proteínas de Ligação ao Cálcio/deficiência , Proteínas de Ligação ao Cálcio/genética , Proteínas de Ligação ao Cálcio/metabolismo , Linhagem Celular , Proteínas Associadas à Distrofina/deficiência , Proteínas Associadas à Distrofina/genética , Hidrocarbonetos Fluorados/farmacologia , Metabolismo dos Lipídeos , Receptores X do Fígado , Macrófagos/efeitos dos fármacos , Proteínas de Membrana/deficiência , Proteínas de Membrana/genética , Proteínas de Membrana/metabolismo , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Endogâmicos DBA , Camundongos Knockout , Complexos Multiproteicos/química , Complexos Multiproteicos/metabolismo , Proteínas Musculares/deficiência , Proteínas Musculares/genética , Proteínas Musculares/metabolismo , Ligação Proteica , Domínios e Motivos de Interação entre Proteínas , Estabilidade Proteica , RNA Mensageiro/genética , RNA Mensageiro/metabolismo , Sulfonamidas/farmacologia , Regulação para CimaRESUMO
HIV-infected patients are at increased risk of developing atherosclerosis, in part due to an altered high density lipoprotein profile exacerbated by down-modulation and impairment of ATP-binding cassette transporter A1 (ABCA1) activity by the HIV-1 protein Nef. However, the mechanisms of this Nef effect remain unknown. Here, we show that Nef interacts with an endoplasmic reticulum chaperone calnexin, which regulates folding and maturation of glycosylated proteins. Nef disrupted interaction between calnexin and ABCA1 but increased affinity and enhanced interaction of calnexin with HIV-1 gp160. The Nef mutant that did not bind to calnexin did not affect the calnexin-ABCA1 interaction. Interaction with calnexin was essential for functionality of ABCA1, as knockdown of calnexin blocked the ABCA1 exit from the endoplasmic reticulum, reduced ABCA1 abundance, and inhibited cholesterol efflux; the same effects were observed after Nef overexpression. However, the effects of calnexin knockdown and Nef on cholesterol efflux were not additive; in fact, the combined effect of these two factors together did not differ significantly from the effect of calnexin knockdown alone. Interestingly, gp160 and ABCA1 interacted with calnexin differently; although gp160 binding to calnexin was dependent on glycosylation, glycosylation was of little importance for the interaction between ABCA1 and calnexin. Thus, Nef regulates the activity of calnexin to stimulate its interaction with gp160 at the expense of ABCA1. This study identifies a mechanism for Nef-dependent inactivation of ABCA1 and dysregulation of cholesterol metabolism.
Assuntos
Transportador 1 de Cassete de Ligação de ATP/metabolismo , Calnexina/metabolismo , Retículo Endoplasmático/metabolismo , Proteína gp160 do Envelope de HIV/metabolismo , Produtos do Gene nef do Vírus da Imunodeficiência Humana/metabolismo , Aterosclerose/metabolismo , Colesterol/metabolismo , Glicosilação , Células HEK293 , HIV-1/metabolismo , Células HeLa , Humanos , Ligação Proteica , Espectrometria de Massas por Ionização por Electrospray , Espectrometria de Massas em TandemRESUMO
Previous studies demonstrated that liver X receptor (LXR) agonists inhibit human immunodeficiency virus (HIV) replication by upregulating cholesterol transporter ATP-binding cassette A1 (ABCA1), suppressing HIV production, and reducing infectivity of produced virions. In this study, we extended these observations by analyzing the effect of the LXR agonist T0901317 [N-[4-(1,1,1,3,3,3-hexafluoro-2-hydroxypropan-2-yl)phenyl]-N-(2,2,2-trifluoroethyl)benzenesulfonamide] on the ongoing HIV infection and investigating the possibility of using LXR agonist for pre-exposure prophylaxis of HIV infection in a humanized mouse model. Pre-exposure of monocyte-derived macrophages to T0901317 reduced susceptibility of these cells to HIV infection in vitro. This protective effect lasted for up to 4 days after treatment termination and correlated with upregulated expression of ABCA1, reduced abundance of lipid rafts, and reduced fusion of the cells with HIV. Pre-exposure of peripheral blood leukocytes to T0901317 provided only a short-term protection against HIV infection. Treatment of HIV-exposed humanized mice with LXR agonist starting 2 weeks postinfection substantially reduced viral load. When eight humanized mice were pretreated with LXR agonist prior to HIV infection, five animals were protected from infection, two had viral load at the limit of detection, and one had viral load significantly reduced relative to mock-treated controls. T0901317 pretreatment also reduced HIV-induced dyslipidemia in infected mice. In conclusion, these results reveal a novel link between LXR stimulation and cell resistance to HIV infection and suggest that LXR agonists may be good candidates for development as anti-HIV agents, in particular for pre-exposure prophylaxis of HIV infection.
Assuntos
Fármacos Anti-HIV/farmacologia , Infecções por HIV/tratamento farmacológico , HIV/efeitos dos fármacos , Hidrocarbonetos Fluorados/farmacologia , Receptores Nucleares Órfãos/agonistas , Receptores Nucleares Órfãos/metabolismo , Sulfonamidas/farmacologia , Transportador 1 de Cassete de Ligação de ATP/metabolismo , Animais , Linhagem Celular , Modelos Animais de Doenças , Células HEK293 , Infecções por HIV/metabolismo , Infecções por HIV/virologia , Humanos , Leucócitos/efeitos dos fármacos , Leucócitos/virologia , Receptores X do Fígado , Macrófagos/efeitos dos fármacos , Macrófagos/virologia , Camundongos , Camundongos Endogâmicos NOD , Regulação para Cima/efeitos dos fármacos , Carga Viral/métodosRESUMO
ABCA12 mutations disrupt the skin barrier and cause harlequin ichthyosis. We previously showed Abca12(-/-) skin has increased glucosylceramide (GlcCer) and correspondingly lower amounts of ceramide (Cer). To examine why loss of ABCA12 leads to accumulation of GlcCer, de novo sphingolipid synthesis was assayed using [(14)C]serine labeling in ex vivo skin cultures. A defect was found in ß-glucocerebrosidase (GCase) processing of newly synthesized GlcCer species. This was not due to a decline in GCase function. Abca12(-/-) epidermis had 5-fold more GCase protein (n = 4, P < 0.01), and a 5-fold increase in GCase activity (n = 3, P < 0.05). As with Abca12(+/+) epidermis, immunostaining in null skin showed a typical interstitial distribution of the GCase protein in the Abca12(-/-) stratum corneum. Hence, we tested whether the block in GlcCer conversion could be circumvented by topically providing GlcCer. This approach restored up to 15% of the lost Cer products of GCase activity in the Abca12(-/-) epidermis. However, this level of barrier ceramide replacement did not significantly reduce trans-epidermal water loss function. Our results indicate loss of ABCA12 function results in a failure of precursor GlcCer substrate to productively interact with an intact GCase enzyme, and they support a model of ABCA12 function that is critical for transporting GlcCer into lamellar bodies.
Assuntos
Transportadores de Cassetes de Ligação de ATP/metabolismo , Epiderme/metabolismo , Glucosilceramidase/metabolismo , Glucosilceramidas/metabolismo , Transportadores de Cassetes de Ligação de ATP/deficiência , Transportadores de Cassetes de Ligação de ATP/genética , Animais , Ceramidas/análise , Ceramidas/metabolismo , Cromatografia em Camada Fina , Epiderme/efeitos dos fármacos , Epiderme/embriologia , Glucosilceramidas/administração & dosagem , Glucosilceramidas/farmacologia , Células HEK293 , Humanos , Immunoblotting , Imuno-Histoquímica , Lipídeos/análise , Lipídeos/química , Camundongos , Camundongos Knockout , Técnicas de Cultura de Órgãos , Reação em Cadeia da Polimerase Via Transcriptase Reversa , Pele/efeitos dos fármacos , Pele/embriologia , Pele/metabolismoRESUMO
HIV infection, through the actions of viral accessory protein Nef, impairs activity of cholesterol transporter ABCA1, inhibiting cholesterol efflux from macrophages and elevating the risk of atherosclerosis. Nef also induces lipid raft formation. In this study, we demonstrate that these activities are tightly linked and affect macrophage function and HIV replication. Nef stimulated lipid raft formation in macrophage cell line RAW 264.7, and lipid rafts were also mobilized in HIV-1-infected human monocyte-derived macrophages. Nef-mediated transfer of cholesterol to lipid rafts competed with the ABCA1-dependent pathway of cholesterol efflux, and pharmacological inhibition of ABCA1 functionality or suppression of ABCA1 expression by RNAi increased Nef-dependent delivery of cholesterol to lipid rafts. Nef reduced cell-surface accessibility of ABCA1 and induced ABCA1 catabolism via the lysosomal pathway. Despite increasing the abundance of lipid rafts, expression of Nef impaired phagocytic functions of macrophages. The infectivity of the virus produced in natural target cells of HIV-1 negatively correlated with the level of ABCA1. These findings demonstrate that Nef-dependent inhibition of ABCA1 is an essential component of the viral replication strategy and underscore the role of ABCA1 as an innate anti-HIV factor.
Assuntos
Transportadores de Cassetes de Ligação de ATP/metabolismo , Colesterol/metabolismo , HIV-1/patogenicidade , Macrófagos/metabolismo , Microdomínios da Membrana/metabolismo , Produtos do Gene nef do Vírus da Imunodeficiência Humana/metabolismo , Transportador 1 de Cassete de Ligação de ATP , Transportadores de Cassetes de Ligação de ATP/genética , Animais , Transporte Biológico , Cloreto de Cálcio/farmacologia , Cloroquina/farmacologia , Infecções por HIV/virologia , HIV-1/fisiologia , Células HeLa , Humanos , Hidrocarbonetos Fluorados/farmacologia , Lisossomos/metabolismo , Macrófagos/efeitos dos fármacos , Macrófagos/virologia , Camundongos , Estabilidade Proteica , Proteólise , Interferência de RNA , Sulfonamidas/farmacologia , Transfecção , Replicação Viral , Produtos do Gene nef do Vírus da Imunodeficiência Humana/genéticaRESUMO
The Weddell seal (Leptonychotes weddellii) thrives in its extreme Antarctic environment. We generated the Weddell seal genome assembly and a high-quality annotation to investigate genome-wide evolutionary pressures that underlie its phenotype and to study genes implicated in hypoxia tolerance and a lipid-based metabolism. Genome-wide analyses included gene family expansion/contraction, positive selection, and diverged sequence (acceleration) compared to other placental mammals, identifying selection in coding and non-coding sequence in five pathways that may shape cardiovascular phenotype. Lipid metabolism as well as hypoxia genes contained more accelerated regions in the Weddell seal compared to genomic background. Top-significant genes were SUMO2 and EP300; both regulate hypoxia inducible factor signaling. Liver expression of four genes with the strongest acceleration signals differ between Weddell seals and a terrestrial mammal, sheep. We also report a high-density lipoprotein-like particle in Weddell seal serum not present in other mammals, including the shallow-diving harbor seal.
Assuntos
Estudo de Associação Genômica Ampla , Genoma , Focas Verdadeiras/genética , Animais , Regiões Antárticas , Regulação da Expressão Gênica/fisiologia , Metabolismo dos Lipídeos , Oxigênio/metabolismo , Filogenia , Especificidade da EspécieRESUMO
ATP-binding cassette transporter A1 (ABCA1)-mediated lipid efflux to apolipoprotein A1 (apoA-I) initiates the biogenesis of high density lipoprotein. Here we show that the Rho guanine nucleotide exchange factors PDZ-RhoGEF and LARG bind to the C terminus of ABCA1 by a PDZ-PDZ interaction and prevent ABCA1 protein degradation by activating RhoA. ABCA1 is a protein with a short half-life, and apoA-I stabilizes ABCA1 protein; however, depletion of PDZ-RhoGEF/LARG by RNA interference suppressed the apoA-I stabilization of ABCA1 protein in human primary fibroblasts. Exogenous PDZ-RhoGEF expression activated RhoA and increased ABCA1 protein levels and cholesterol efflux activity. Likewise, forced expression of a constitutively active RhoA mutant significantly increased ABCA1 protein levels, whereas a dominant negative RhoA mutant decreased them. The constitutively active RhoA retarded ABCA1 degradation, thus accounting for its ability to increase ABCA1 protein. Moreover, stimulation with apoA-I transiently activated RhoA, and the pharmacological inhibition of RhoA or the dominant negative RhoA blocked the ability of apoA-I to stabilize ABCA1. Finally, depletion of RhoA or RhoGEFs/RhoA reduces the cholesterol efflux when transcriptional regulation via PPARgamma is eliminated. Taken together, our results have identified a novel physical and functional interaction between ABCA1 and PDZ-RhoGEF/LARG, which activates RhoA, resulting in ABCA1 stabilization and cholesterol efflux activity.
Assuntos
Transportadores de Cassetes de Ligação de ATP/metabolismo , Colesterol/metabolismo , Fibroblastos/metabolismo , Fatores de Troca do Nucleotídeo Guanina/metabolismo , Proteína rhoA de Ligação ao GTP/metabolismo , Transportador 1 de Cassete de Ligação de ATP , Transportadores de Cassetes de Ligação de ATP/genética , Apolipoproteína A-I/genética , Apolipoproteína A-I/metabolismo , Transporte Biológico/fisiologia , Linhagem Celular , Colesterol/genética , Genes Dominantes , Fatores de Troca do Nucleotídeo Guanina/genética , Humanos , Lipoproteínas HDL/genética , Lipoproteínas HDL/metabolismo , Mutação , Domínios PDZ , PPAR gama/genética , PPAR gama/metabolismo , Estabilidade Proteica , Fatores de Troca de Nucleotídeo Guanina Rho , Proteína rhoA de Ligação ao GTP/genéticaRESUMO
Human immunodeficiency virus (HIV) infection and subsequent antiretroviral therapy have been associated with an increased incidence of dyslipidemia and cardiovascular disease and has been shown to suppress cholesterol efflux from virus-infected macrophages by inducing Nef-dependent down-regulation of adenosine triphosphate-binding cassette transporter A1 (ABCA1). Here, the simian immunodeficiency virus (SIV)-infected macaque model was used to examine the consequences and mechanisms involved. SIV infection drove a significant remodeling of high-density lipoprotein profiles, suggesting that systemic inhibition of the ABCA1-dependent reverse cholesterol transport pathway occurred. The ABCA1 cholesterol transporter was significantly down-regulated in the livers of the SIV-infected macaques, and the viral protein Nef could be detected in the livers as well as in the plasma of infected animals. Extracellular myristoylated HIV Nef inhibited cholesterol efflux from macrophages and hepatocytes. Moreover, serum samples from SIV-infected macaques also suppressed cholesterol efflux in a Nef-dependent fashion. These results indicate that SIV infection is a significant contributor to primary dyslipidemia, likely through the ability of Nef to suppress ABCA1-dependent reverse cholesterol transport.
Assuntos
Transportadores de Cassetes de Ligação de ATP/antagonistas & inibidores , Colesterol/metabolismo , Dislipidemias/sangue , Síndrome de Imunodeficiência Adquirida dos Símios/complicações , Vírus da Imunodeficiência Símia/patogenicidade , Produtos do Gene nef do Vírus da Imunodeficiência Humana/sangue , Transportador 1 de Cassete de Ligação de ATP , Animais , Células Cultivadas , Hepatócitos/metabolismo , Fígado/química , Fígado/enzimologia , Macaca mulatta , Macrófagos/metabolismo , Síndrome de Imunodeficiência Adquirida dos Símios/fisiopatologia , Produtos do Gene nef do Vírus da Imunodeficiência Humana/análiseRESUMO
The ATP binding cassette subfamily A member 7 (ABCA7) gene is one of the significant susceptibility loci for Alzheimer's disease (AD). Furthermore, ABCA7 loss of function variants resulting from premature termination codon in the gene are associated with increased risk for AD. ABCA7 belongs to the ABC transporter family, which mediates the transport of diverse metabolites across the cell membrane. ABCA7 is also involved in modulating immune responses. Because the immune system and lipid metabolism causatively engage in the pathogenesis of AD, we investigated how ABCA7 haplodeficiency modulates the metabolic profile in mouse brains during acute immune response using a metabolomics approach through LC/Q-TOF-MS. Peripheral lipopolysaccharide (LPS) stimulation substantially influenced the metabolite content in the cortex, however, the effect on metabolic profiles in Abca7 heterozygous knockout mice (Abca7 ±) was modest compared to that in the control wild-type mice. Weighted gene co-expression network analysis (WGCNA) of the metabolomics dataset identified two modules influenced by LPS administration and ABCA7 haplodeficiency, in which glycerophospholipid metabolism, linoleic acid metabolism, and α-linolenic acid metabolism were identified as major pathways. Consistent with these findings, we also found that LPS stimulation increased the brain levels of eicosapentaenoic acid, oleic acid, and palmitic acid in Abca7 ± mice, but not control mice. Together, our results indicate that ABCA7 is involved in the crosstalk between fatty acid metabolism and inflammation in the brain, and disturbances in these pathways may contribute to the risk for AD.
RESUMO
Chronic HIV infection may exacerbate atherosclerotic vascular disease, which at advanced stages presents as necrotic plaques rich in crystalline cholesterol. Such lesions can catastrophically rupture precipitating myocardial infarct and stroke, now important causes of mortality in those living with HIV. However, in this population little is known about plaque structure relative to crystalline content and its chemical composition. Here, we first interrogated plaque crystal structure and composition in atherosclerotic SIV-infected macaques using non-linear optical microscopy. By stimulated Raman scattering and second harmonic generation approaches both amorphous and crystalline plaque lipid was detected and the crystal spectral profile indicated a cholesterol ester (CE) dominated composition. Versus controls, SIV+ samples had a greater number of cholesterol crystals (CCs), with the difference, in part, accounted for by crystals of a smaller length. Given the ester finding, we profiled HIV+ plaques and also observed a CE crystalline spectral signature. We further profiled plaques from Ldlr-/- mice fed a high fat diet, and likewise, found CE-dominate crystals. Finally, macrophage exposure to CCs or AcLDL induced auto-fluorescent puncta that co-stained with the LC3B autophagy sensor. In aggregate, we show that atheromatous plaques from mice, macaques and humans, display necrotic cores dominated by esterified CCs, and that plaque macrophages may induce autophagic vesicle formation upon encountering CCs. These findings help inform our knowledge of plaque core lipid evolution and how the process may incite systemic inflammation.
Assuntos
Ésteres do Colesterol/análise , Infecções por HIV/patologia , Placa Aterosclerótica/patologia , Síndrome de Imunodeficiência Adquirida dos Símios/patologia , Animais , HIV/isolamento & purificação , Infecções por HIV/complicações , Macaca , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Imagem Óptica , Placa Aterosclerótica/complicações , Células RAW 264.7 , Síndrome de Imunodeficiência Adquirida dos Símios/complicações , Vírus da Imunodeficiência Símia/isolamento & purificaçãoRESUMO
HIV-1 infection and antiretroviral therapy are associated with a dyslipidemia marked by low levels of high-density lipoprotein and increased cardiovascular disease, but it is unclear whether virion replication plays a causative role in these changes. The HIV-1 Nef protein can impair ATP cassette binding transporter A1 (ABCA1) cholesterol efflux from macrophages, a potentially pro-atherosclerotic effect. This viral inhibition of efflux was correlated with a direct interaction between ABCA1 and Nef. Here, we defined the ABCA1 domain required for the Nef-ABCA1 protein-protein interaction and determined whether this interaction mediates the ability of Nef to downregulate ABCA1. Nef expressed in HEK 293 cells strongly inhibited ABCA1 efflux and protein levels but did not alter levels of cMIR, another transmembrane protein. Analysis of a panel of ABCA1 C-terminal mutants showed Nef binding required the ABCA1 C-terminal amino acids between positions 2225 and 2231. However, the binding of Nef to ABCA1 was not required for inhibition because the C-terminal ABCA1 mutants that did not bind Nef were still downregulated by Nef. Given this discordance, the mechanism of downregulation was investigated and was found to involve the acceleration of ABCA1 protein degradation but did not to depend upon the ABCA1 PEST sequence, which mediates the calpain proteolysis of ABCA1. Furthermore, it did not associate with a Nef-dependent induction of signaling through the unfolded protein response but was significantly dependent upon proteasomal function and could act on an ABCA1 mutant that fails to exit the endoplasmic reticulum. In summary, we show that Nef downregulates ABCA1 function by a post-translational mechanism that stimulates ABCA1 degradation but does not require the ability of Nef to bind ABCA1.
Assuntos
Transportadores de Cassetes de Ligação de ATP/genética , Mutação , Produtos do Gene nef do Vírus da Imunodeficiência Humana/metabolismo , Transportador 1 de Cassete de Ligação de ATP , Transportadores de Cassetes de Ligação de ATP/metabolismo , Animais , Transporte Biológico/genética , Linhagem Celular , Regulação para Baixo , Lipoproteínas HDL/genética , Lipoproteínas HDL/metabolismo , Macrófagos/metabolismo , Proteínas de Membrana Transportadoras/genética , Proteínas de Membrana Transportadoras/metabolismo , Camundongos , Resposta a Proteínas não DobradasRESUMO
We previously reported that the endogenous ATP-binding cassette transporter (ABC)A7 strongly associates with phagocytic function rather than biogenesis of high-density lipoprotein (HDL), being regulated by sterol-regulatory element binding protein (SREBP)2. Phagocytic activity was found enhanced by apolipoprotein (apo)A-I and apoA-II more than twice the maximum in J774 and mouse peritoneal macrophages. Therefore we investigated the molecular basis of this reaction in association with the function of ABCA7. Similar to ABCA1, ABCA7 was degraded, likely by calpain, and apoA-I and apoA-II stabilize ABCA7 against degradation. Cell surface biotinylation experiments demonstrated that endogenous ABCA7 predominantly resides on the cell surface and that the apolipoproteins increase the surface ABCA7. The increase of phagocytosis by apolipoproteins was retained in the J774 cells treated with ABCA1 siRNA and in the peritoneal macrophages from ABCA1-knockout mice, but it was abolished in the J774 cells treated with ABCA7 siRNA and in the peritoneal macrophages from ABCA7-knockout mice. Phagocytosis was decreased in the cells in the peritoneal cavity of the ABCA7-knockout mouse compared with the wild-type control. We thus concluded that extracellular helical apolipoproteins augment ABCA7-associated phagocytosis by stabilizing ABCA7. The results demonstrated direct enhancement of the host defense system by HDL components.
Assuntos
Transportadores de Cassetes de Ligação de ATP/metabolismo , Apolipoproteínas , Lipoproteínas HDL , Fagocitose/fisiologia , Transportador 1 de Cassete de Ligação de ATP , Transportadores de Cassetes de Ligação de ATP/genética , Animais , Apolipoproteína A-I/farmacologia , Apolipoproteína A-II/farmacologia , Apolipoproteínas/química , Apolipoproteínas/metabolismo , Linhagem Celular , Fibroblastos/citologia , Fibroblastos/efeitos dos fármacos , Fibroblastos/metabolismo , Humanos , Lipoproteínas HDL/química , Lipoproteínas HDL/metabolismo , Macrófagos Peritoneais/citologia , Macrófagos Peritoneais/efeitos dos fármacos , Macrófagos Peritoneais/metabolismo , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Knockout , RNA Interferente Pequeno/genética , RNA Interferente Pequeno/metabolismo , Staphylococcus aureus/metabolismoRESUMO
Elevated sphingolipids have been associated with increased cardiovascular disease. Conversely, atherosclerosis is reduced in mice by blocking de novo synthesis of sphingolipids catalyzed by serine palmitoyltransferase (SPT). The SPT enzyme is composed of the SPTLC1 and -2 subunits, and here we describe a novel protein-protein interaction between SPTLC1 and the PDZ protein Par3 (partitioning defective protein 3). Mammalian SPTLC1 orthologs have a highly conserved C terminus that conforms to a type II PDZ protein interaction motif, and by screening PDZ domain protein arrays with an SPTLC1 C-terminal peptide, we found it bound the third PDZ domain of Par3. Overlay and immunoprecipitation assays confirmed this interaction and indicate Par3 is able to associate with the SPTLC1/2 holoenzyme by binding the C-terminal SPTLC1 PDZ motif. The physiologic existence of the SPTLC1/2-Par3 complex was detected in mouse liver and macrophages, and short interfering RNA inhibition of Par3 in human THP-1 monocytes significantly reduced SPT activity and de novo ceramide synthesis by nearly 40%. Given monocyte recruitment into inflamed vessels is thought to promote atherosclerosis, and because Par3 and sphingolipids have been associated with polarized cell migration, we tested whether the ability of THP-1 monocytes to migrate toward MCP-1 (monocyte chemoattractant protein 1) depended upon Par3 and SPTLC1 expression. Knockdown of Par3 significantly reduced MCP1-induced chemotaxis of THP-1 monocytes, as did knockdown of SPTLC1, and this Par3 effect depended upon SPT activity and was blunted by ceramide treatment. In conclusion, protein arrays were used to identify a novel SPTLC1-Par3 interaction that associates with increased monocyte serine palmitoyltransferase activity and chemotaxis toward inflammatory signals.
Assuntos
Proteínas de Ciclo Celular/metabolismo , Quimiotaxia , Proteínas de Membrana/metabolismo , Monócitos/citologia , Serina C-Palmitoiltransferase/metabolismo , Proteínas Adaptadoras de Transdução de Sinal , Motivos de Aminoácidos , Sequência de Aminoácidos , Linhagem Celular , Movimento Celular , Humanos , Inflamação , Dados de Sequência Molecular , Monócitos/metabolismo , Estrutura Terciária de Proteína , Homologia de Sequência de Aminoácidos , Esfingolipídeos/metabolismoRESUMO
PURPOSE: Polyamines are essential for the sustained proliferation and biomass required by tumor cells. Bis-alkylated polyamine analogs are nonfunctional competitors of natural polyamines. Of these, PG-11047, a second-generation unsaturated analog of the polyamine spermine, has demonstrated anticancer activity in cell lines and animal models of multiple cancer types. This report describes the first phase I clinical trial to investigate PG-11047 in patients with advanced refractory metastatic solid tumors. METHODS: Forty-six patients were treated with 60-min intravenous infusions of PG-11047 using a 28-day dosing cycle with treatments on days 1, 8, and 15. Doses ranged from 50 to 750 mg. The treatment period consisted of at least two cycles. RESULTS: The maximum tolerated dose of PG-11047 administered at this dosing schedule was 610 mg. Dose-limiting toxicities (DLT) were mainly gastrointestinal, including oral/anal mucositis and diarrhea; other DLTs included one case each of angioedema and a grade 3 alanine aminotransferase (ALT) increase. The most common adverse effects were fatigue and anorexia. Stable disease was documented in 30% of patients. CONCLUSION: Results of this phase I trial suggest that PG-11047 can be safely administered to patients on the once weekly dosing schedule described. The manageable toxicity profile and high MTD determination provide a safety profile for further clinical studies, including those in combination with current chemotherapeutic agents.
Assuntos
Neoplasias/tratamento farmacológico , Espermina/análogos & derivados , Adulto , Idoso , Idoso de 80 Anos ou mais , Relação Dose-Resposta a Droga , Feminino , Seguimentos , Humanos , Masculino , Pessoa de Meia-Idade , Neoplasias/patologia , Prognóstico , Espermina/administração & dosagem , Espermina/farmacocinética , Distribuição TecidualRESUMO
People living with HIV on antiretroviral treatment have significantly improved longevity, but as a result may also face increasing multimorbidity due to aging and long-term medication use. Thus, care needs for this population have evolved to require a chronic disease management approach in which self-management plays a central role. Here we highlight the importance of expanding self-management support options for people living with HIV, and discuss strategies for implementing and evaluating self-management interventions, outlining potential opportunities, challenges and solutions. We contend that standardized programs such as those offered through the Self-Management Resource Centre provide a rich opportunity to build the evidence base regarding the potential effectiveness of self-management support among people living with HIV. Thus we recommend enhancing self-management support through meaningful community-level collaboration with people with lived experience, careful assessment of process and outcome factors including who does not participate and why, attention to stigma and the specific needs of HIV priority groups, and consideration of how to extend engagement with services to address social and material needs beyond self-management program participation. We hope this reflection will serve as an aide for researchers and program managers to improve the array of evidence-based self-management support options available to people living with HIV.