RESUMO
Long intergenic noncoding RNAs (lincRNAs) have emerged as key regulators of cellular functions and physiology. Yet functional lincRNAs often have low, context-specific and tissue-specific expression. We hypothesized that many human monocyte and adipose lincRNAs would be absent in current public annotations due to lincRNA tissue specificity, modest sequencing depth in public data, limitations of transcriptome assembly algorithms, and lack of dynamic physiological contexts. Deep RNA sequencing (RNA-Seq) was performed in peripheral blood CD14+ monocytes (monocytes; average ~247 million reads per sample) and adipose tissue (average ~378 million reads per sample) collected before and after human experimental endotoxemia, an in vivo inflammatory stress, to identify tissue-specific and clinically relevant lincRNAs. Using a stringent filtering pipeline, we identified 109 unannotated lincRNAs in monocytes and 270 unannotated lincRNAs in adipose. Most unannotated lincRNAs are not conserved in rodents and are tissue specific, while many have features of regulated expression and are enriched in transposable elements. Specific subsets have enhancer RNA characteristics or are expressed only during inflammatory stress. A subset of unannotated lincRNAs was validated and replicated for their presence and inflammatory induction in independent human samples and for their monocyte and adipocyte origins. Through interrogation of public genome-wide association data, we also found evidence of specific disease association for selective unannotated lincRNAs. Our findings highlight the critical need to perform deep RNA-Seq in a cell-, tissue-, and context-specific manner to annotate the full repertoire of human lincRNAs for a complete understanding of lincRNA roles in dynamic cell functions and in human disease.
Assuntos
Inflamação/metabolismo , Receptores de Lipopolissacarídeos/metabolismo , RNA Longo não Codificante/genética , Tecido Adiposo/metabolismo , Adolescente , Adulto , Elementos de DNA Transponíveis/genética , Endotoxemia/genética , Endotoxemia/metabolismo , Perfilação da Expressão Gênica/métodos , Estudo de Associação Genômica Ampla , Humanos , Inflamação/genética , Masculino , Pessoa de Meia-Idade , Monócitos/metabolismo , Polimorfismo de Nucleotídeo Único/genética , Adulto JovemRESUMO
RATIONALE: An efficient and reproducible source of genotype-specific human macrophages is essential for study of human macrophage biology and related diseases. OBJECTIVE: To perform integrated functional and transcriptome analyses of human induced pluripotent stem cell-derived macrophages (IPSDMs) and their isogenic human peripheral blood mononuclear cell-derived macrophage (HMDM) counterparts and assess the application of IPSDM in modeling macrophage polarization and Mendelian disease. METHODS AND RESULTS: We developed an efficient protocol for differentiation of IPSDM, which expressed macrophage-specific markers and took up modified lipoproteins in a similar manner to HMDM. Like HMDM, IPSDM revealed reduction in phagocytosis, increase in cholesterol efflux capacity and characteristic secretion of inflammatory cytokines in response to M1 (lipopolysaccharide+interferon-γ) activation. RNA-Seq revealed that nonpolarized (M0) as well as M1 or M2 (interleukin-4) polarized IPSDM shared transcriptomic profiles with their isogenic HMDM counterparts while also revealing novel markers of macrophage polarization. Relative to IPSDM and HMDM of control individuals, patterns of defective cholesterol efflux to apolipoprotein A-I and high-density lipoprotein-3 were qualitatively and quantitatively similar in IPSDM and HMDM of patients with Tangier disease, an autosomal recessive disorder because of mutations in ATP-binding cassette transporter AI. Tangier disease-IPSDM also revealed novel defects of enhanced proinflammatory response to lipopolysaccharide stimulus. CONCLUSIONS: Our protocol-derived IPSDM are comparable with HMDM at phenotypic, functional, and transcriptomic levels. Tangier disease-IPSDM recapitulated hallmark features observed in HMDM and revealed novel inflammatory phenotypes. IPSDMs provide a powerful tool for study of macrophage-specific function in human genetic disorders as well as molecular studies of human macrophage activation and polarization.
Assuntos
Técnicas de Cultura de Células , Células-Tronco Pluripotentes Induzidas/citologia , Macrófagos/metabolismo , Doença de Tangier/patologia , Transcriptoma , Transportador 1 de Cassete de Ligação de ATP/deficiência , Transportador 1 de Cassete de Ligação de ATP/genética , Transportador 1 de Cassete de Ligação de ATP/fisiologia , Adulto , Idoso , Animais , Antígenos de Diferenciação/análise , Sequência de Bases , Diferenciação Celular , Células Cultivadas , Colesterol/metabolismo , Corpos Embrioides/citologia , Feminino , Genótipo , Humanos , Células-Tronco Pluripotentes Induzidas/metabolismo , Inflamação , Interferon gama/farmacologia , Lipopolissacarídeos/farmacologia , Ativação de Macrófagos/efeitos dos fármacos , Macrófagos/citologia , Macrófagos/efeitos dos fármacos , Masculino , Camundongos , Camundongos Knockout , Dados de Sequência Molecular , Fagocitose , Fenótipo , RNA Mensageiro/genética , Alinhamento de Sequência , Homologia de Sequência do Ácido Nucleico , Doença de Tangier/genética , Doença de Tangier/metabolismo , Adulto JovemRESUMO
BACKGROUND: Chronic inflammation may contribute to insulin resistance (IR), metabolic syndrome and atherosclerosis although evidence of causality is lacking in humans. We hypothesized that very low-dose experimental endotoxemia would induce adipose tissue inflammation and systemic IR during a low-grade but asymptomatic inflammatory response and thus provide an experimental model for future tests of pharmacologic and genomic modulation of cardio-metabolic traits in humans. METHODS: Ten healthy, human volunteers (50% male, 90% Caucasian, mean age 22.7 ± 3.8) were randomized in a double-masked, placebo-controlled, crossover study to separate 36-hour inpatient visits (placebo versus intravenous-LPS 0.6 ng/kg). We measured clinical symptoms via the McGill pain questionnaire and serial vital signs. Plasma and serum were collected for measurement of cytokines, C-reactive protein, insulin and glucose, serial whole blood & subcutaneous adipose tissue mRNA expression were measured by real-time PCR. HOMA-IR, a well-validated measure of IR was calculated to estimate insulin resistance, and frequently sampled intravenous glucose tolerance testing (FSIGTT) was performed to confirm an insulin resistant state. We performed ANOVA and within subject ANOVA to understand the differences in cytokines, adipose tissue inflammation and IR before and after LPS or placebo. RESULTS: There was no significant difference between placebo and LPS in clinical responses of symptom scores, body temperature or heart rate. However, low-dose endotoxemia induced a rapid and transient 25-fold induction of plasma TNF-alpha and 100-fold increase in plasma IL-6 (Figure 1B) (p < 0.001 for both) both peaking at two hours, followed by modest inflammation in adipose tissue with increases in mRNA levels of several inflammatory genes known to modulate adipose and systemic insulin resistance. Adipose tissue mRNA levels of IL-6 (peak 6-fold, ANOVA F = 27.5, p < 0.001) and TNF-alpha (peak 1.8-fold, F = 2.9, p = 0.01) increased with MCP-1 (peak 10-fold, F = 5.6, p < 0.01) and fractalkine (CX3CL1) (peak 15-fold, F = 13.3, p < 0.001). Finally, HOMA-IR was 32% higher following LPS compared to placebo (p < 0.01) and insulin sensitivity declined by 21% following LPS compared to placebo (p < 0.05). CONCLUSIONS: We present a low dose human endotoxemia model of inflammation which induces adipose tissue inflammation and systemic insulin resistance in the absence of overt clinical response. Such a model has the potential for broad and safe application in the study of novel therapeutics and genomic influences in cardio-metabolic disease.
Assuntos
Inflamação/patologia , Síndrome Metabólica/patologia , Adulto , Estudos Cross-Over , Método Duplo-Cego , Feminino , Humanos , Inflamação/sangue , Inflamação/metabolismo , Resistência à Insulina , Masculino , Síndrome Metabólica/sangue , Síndrome Metabólica/metabolismo , PlacebosRESUMO
BACKGROUND: Adipose harbors a large depot of free cholesterol. However, a role for adipose in cholesterol lipidation of high-density lipoprotein (HDL) in vivo is not established. We present the first evidence that adipocytes support transfer of cholesterol to HDL in vivo as well as in vitro and implicate ATP-binding cassette subfamily A member 1 (ABCA1) and scavenger receptor class B type I (SR-BI), but not ATP-binding cassette subfamily G member 1 (ABCG1), cholesterol transporters in this process. METHODS AND RESULTS: Cholesterol efflux from wild-type, ABCA1(-/-), SR-BI(-/-), and ABCG1(-/-) adipocytes to apolipoprotein A-I (apoA-I) and HDL3 were measured in vitro. 3T3L1 adipocytes, labeled with (3)H-cholesterol, were injected intraperitoneally into wild-type, apoA-I transgenic, and apoA-I(-/-) mice, and tracer movement onto plasma HDL was monitored. Identical studies were performed with labeled wild-type, ABCA1(-/-), or SR-BI(-/-) mouse embryonic fibroblast adipocytes. The effect of tumor necrosis factor-alpha on transporter expression and cholesterol efflux was monitored during adipocyte differentiation. Cholesterol efflux to apoA-I and HDL3 was impaired in ABCA1(-/-) and SR-BI(-/-) adipocytes, respectively, with no effect observed in ABCG1(-/-) adipocytes. Intraperitoneal injection of labeled 3T3L1 adipocytes resulted in increased HDL-associated (3)H-cholesterol in apoA-I transgenic mice but reduced levels in apoA-I(-/-) animals. Intraperitoneal injection of labeled ABCA1(-/-) or SR-BI(-/-) adipocytes reduced plasma counts relative to their respective controls. Tumor necrosis factor-alpha reduced both ABCA1 and SR-BI expression and impaired cholesterol efflux from partially differentiated adipocytes. CONCLUSIONS: These data suggest a novel metabolic function of adipocytes in promoting cholesterol transfer to HDL in vivo and implicate adipocyte SR-BI and ABCA1, but not ABCG1, in this process. Furthermore, adipocyte modulation of HDL may be impaired in adipose inflammatory disease states such as type 2 diabetes mellitus.
Assuntos
Adipócitos/metabolismo , HDL-Colesterol/metabolismo , Colesterol/metabolismo , Células 3T3-L1 , Transportador 1 de Cassete de Ligação de ATP , Membro 1 da Subfamília G de Transportadores de Cassetes de Ligação de ATP , Transportadores de Cassetes de Ligação de ATP/genética , Transportadores de Cassetes de Ligação de ATP/metabolismo , Adipócitos/citologia , Animais , Apolipoproteína A-I/genética , Apolipoproteína A-I/metabolismo , Diferenciação Celular , Células Cultivadas , Humanos , Inflamação/metabolismo , Lipoproteínas/genética , Lipoproteínas/metabolismo , Camundongos , Camundongos Knockout , Camundongos Transgênicos , Modelos Animais , Receptores Depuradores Classe B/genética , Receptores Depuradores Classe B/metabolismo , Fator de Necrose Tumoral alfa/metabolismoRESUMO
Recent reports demonstrate T-cell infiltration of adipose tissue in early obesity. We hypothesized that interferon (IFN) gamma, a major T-cell inflammatory cytokine, would attenuate human adipocyte functions and sought to establish signaling mechanisms. Differentiated human adipocytes were treated with IFNgamma +/- pharmacological inhibitors prior to insulin stimulation. [(3)H]Glucose uptake and AKT phosphorylation were assessed as markers of insulin sensitivity. IFNgamma induced sustained loss of insulin-stimulated glucose uptake in human adipocytes, coincident with reduced Akt phosphorylation and down-regulation of the insulin receptor, insulin receptor substrate-1, and GLUT4. Loss of adipocyte triglyceride storage was observed with IFNgamma co-incident with reduced expression of peroxisome proliferator-activated receptor gamma, adiponectin, perilipin, fatty acid synthase, and lipoprotein lipase. Treatment with IFNgamma also blocked differentiation of pre-adipocytes to the mature phenotype. IFNgamma-induced robust STAT1 phosphorylation and SOCS1 mRNA expression, with modest, transient STAT3 phosphorylation and SOCS3 induction. Preincubation with a non-selective JAK inhibitor restored glucose uptake and Akt phosphorylation while completely reversing IFNgamma suppression of adipogenic mRNAs and adipocyte differentiation. Specific inhibition of JAK2 or JAK3 failed to block IFNgamma effects suggesting a predominant role for JAK1-STAT1. We demonstrate that IFNgamma attenuates insulin sensitivity and suppresses differentiation in human adipocytes, an effect most likely mediated via sustained JAK-STAT1 pathway activation.
Assuntos
Adipócitos/metabolismo , Diferenciação Celular , Insulina/metabolismo , Interferon gama/farmacologia , Janus Quinase 1/metabolismo , Fator de Transcrição STAT1/metabolismo , Triglicerídeos/metabolismo , Western Blotting , Glucose/metabolismo , Transportador de Glucose Tipo 4/metabolismo , Humanos , Hipoglicemiantes/metabolismo , Proteínas Substratos do Receptor de Insulina/metabolismo , Resistência à Insulina , Metabolismo dos Lipídeos , PPAR gama/metabolismo , Fosforilação , Proteínas Proto-Oncogênicas c-akt/metabolismo , RNA Mensageiro/genética , RNA Mensageiro/metabolismo , Proteínas Recombinantes , Reação em Cadeia da Polimerase Via Transcriptase Reversa , Transdução de SinaisRESUMO
BACKGROUND: Inflammation is proposed to impair reverse cholesterol transport (RCT), a major atheroprotective function of high-density lipoprotein (HDL). The present study presents the first integrated functional evidence that inflammation retards numerous components of RCT. METHODS AND RESULTS: We used subacute endotoxemia in the rodent macrophage-to-feces RCT model to assess the effects of inflammation on RCT in vivo and performed proof of concept experimental endotoxemia studies in humans. Endotoxemia (3 mg/kg SC) reduced (3)H-cholesterol movement from macrophage to plasma and (3)H-cholesterol associated with HDL fractions. At 48 hours, bile and fecal counts were markedly reduced consistent with downregulation of hepatic expression of ABCG5, ABCG8, and ABCB11 biliary transporters. Low-dose lipopolysaccharide (0.3 mg/kg SC) also reduced bile and fecal counts, as well as expression of biliary transporters, but in the absence of effects on plasma or liver counts. In vitro, lipopolysaccharide impaired (3)H-cholesterol efflux from human macrophages to apolipoprotein A-I and serum coincident with reduced expression of the cholesterol transporter ABCA1. During human (3 ng/kg; n=20) and murine endotoxemia (3 mg/kg SC), ex vivo macrophage cholesterol efflux to acute phase HDL was attenuated. CONCLUSIONS: We provide the first in vivo evidence that inflammation impairs RCT at multiple steps in the RCT pathway, particularly cholesterol flux through liver to bile and feces. Attenuation of RCT and HDL efflux function, independent of HDL cholesterol levels, may contribute to atherosclerosis in chronic inflammatory states including obesity, metabolic syndrome, and type 2 diabetes.
Assuntos
Colesterol/metabolismo , Endotoxemia/patologia , Adolescente , Adulto , Animais , Aterosclerose/metabolismo , Aterosclerose/patologia , Aterosclerose/prevenção & controle , Transporte Biológico Ativo/fisiologia , Linhagem Celular , Endotoxemia/metabolismo , Feminino , Humanos , Inflamação/metabolismo , Inflamação/patologia , Lipoproteínas HDL/metabolismo , Macrófagos/metabolismo , Macrófagos/patologia , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Adulto JovemRESUMO
CONTEXT: Chronic inflammation converges in type 2 diabetes and atherosclerosis. Modulation of adipokine signaling by innate immunity in humans is of considerable interest given the role of adipokines in insulin resistance and atherosclerosis. OBJECTIVE: The aim of the study was to examine effects of low-grade endotoxemia, a model of human inflammation, on adipokines in vivo. DESIGN/SETTING: An open-label, placebo-controlled, fixed-sequence clinical study was conducted at a General Clinical Research Center. PATIENTS: There were 20 healthy male (50%) and female volunteers aged 18-40 yr. INTERVENTION: Serial blood sampling and adipose biopsies were performed for 24 h before and after iv bolus endotoxin [lipopolysaccharide (LPS), 3 ng/kg]. MAIN OUTCOME MEASURES: We measured plasma leptin, adiponectin, resistin, soluble leptin receptor, cytokines, insulin, and glucose; distribution of adiponectin among multimeric complexes; whole blood, monocyte and adipose mRNA for adipokines and their receptors. RESULTS: LPS induced fever, blood, and adipose TNF and IL-6 and increased homeostasis model assessment of insulin resistance. These were associated with increases in plasma leptin (from 4.1 +/- 1.1 to 6.1 +/- 1.9 ng/ml in men; 21.1 +/- 4.4 to 27.4 +/- 4.7 ng/ml in women; P < 0.005), doubling of the leptin:soluble leptin receptor ratio, and marked induction of whole blood resistin mRNA (13.7 +/- 7.3-fold; P < 0.001) and plasma resistin (8.5 +/- 2.75 to 43.2 +/- 15.3 ng/ml; P < 0.001). Although total adiponectin levels and low and high molecular weight adiponectin complexes were unaltered by LPS treatment, whole blood mRNA for adiponectin receptors 1 (49%; P < 0.005) and 2 (65%; P < 0.001) was suppressed. CONCLUSIONS: Modulation of adipokine signaling may contribute to the insulin resistant, atherogenic state associated with human inflammatory syndromes. Targeting of individual adipokines or their upstream regulation may prove effective in preventing acute and chronic inflammation-related metabolic complications.
Assuntos
Endotoxemia/imunologia , Endotoxemia/metabolismo , Sistema Imunitário/imunologia , Sistema Imunitário/metabolismo , Hormônios Peptídicos/sangue , Adiponectina/sangue , Adiponectina/genética , Adulto , Glicemia/metabolismo , Citocinas/sangue , Citocinas/genética , Endotoxemia/induzido quimicamente , Feminino , Humanos , Insulina/sangue , Leptina/sangue , Leptina/genética , Lipopolissacarídeos/administração & dosagem , Masculino , Hormônios Peptídicos/genética , Placebos , RNA Mensageiro/metabolismo , Receptores de Superfície Celular/sangue , Receptores de Superfície Celular/genética , Receptores para Leptina , Resistina/sangue , Resistina/genética , Transdução de Sinais/imunologiaRESUMO
OBJECTIVE: The purpose of the study was to explore the impact of dual targeting of C-C motif chemokine receptor-2 (CCR2) and fractalkine receptor (CX3CR1) on the metabolic and inflammatory consequences of obesity induced by a high-fat diet (HFD). METHODS: C57BL/6J wild-type, Cx3cr1-/- , Ccr2-/- , and Cx3cr1-/- Ccr2-/- double-knockout male and female mice were fed a 45% HFD for up to 25 weeks starting at 12 weeks of age. RESULTS: All groups gained weight at a similar rate and developed a similar degree of adiposity, hyperglycemia, glucose intolerance, and impairment of insulin sensitivity in response to HFD. As expected, the circulating monocyte count was decreased in Ccr2-/- and Cx3cr1-/- Ccr2-/- mice but not in Cx3cr1-/- mice. Flow cytometric analysis of perigonadal adipose tissue of male, but not female, mice revealed trends to lower CD11c+MGL1- M1-like macrophages and higher CD11c-MGL1+ M2-like macrophages as a percentage of CD45+F4/80+CD11b+ macrophages in Cx3cr1-/- Ccr2-/- mice versus wild-type mice, suggesting reduced adipose tissue macrophage activation. In contrast, single knockout of Ccr2 or Cx3cr1 did not differ in their adipose macrophage phenotypes. CONCLUSIONS: Although CCR2 and CX3CR1 may synergistically impact inflammatory phenotypes, their joint deficiency did not influence the metabolic effects of a 45% HFD-induced obesity in these model conditions.
Assuntos
Receptor 1 de Quimiocina CX3C/metabolismo , Dieta Hiperlipídica/efeitos adversos , Receptores CCR2/metabolismo , Aumento de Peso , Animais , Composição Corporal , Receptor 1 de Quimiocina CX3C/genética , Feminino , Intolerância à Glucose/etiologia , Intolerância à Glucose/genética , Inflamação , Insulina/metabolismo , Resistência à Insulina , Secreção de Insulina , Macrófagos/metabolismo , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Knockout , Obesidade/etiologia , Obesidade/genética , Receptores CCR2/genéticaRESUMO
BACKGROUND: Sustained and dysfunctional macrophage activation promotes inflammatory cardiometabolic disorders, but the role of long intergenic noncoding RNA (lincRNA) in human macrophage activation and cardiometabolic disorders is poorly defined. Through transcriptomics, bioinformatics, and selective functional studies, we sought to elucidate the lincRNA landscape of human macrophages. METHODS AND RESULTS: We used deep RNA sequencing to assemble the lincRNA transcriptome of human monocyte-derived macrophages at rest and following stimulation with lipopolysaccharide and IFN-γ (interferon γ) for M1 activation and IL-4 (interleukin 4) for M2 activation. Through de novo assembly, we identified 2766 macrophage lincRNAs, including 861 that were previously unannotated. The majority (≈85%) was nonsyntenic or was syntenic but not annotated as expressed in mouse. Many macrophage lincRNAs demonstrated tissue-enriched transcription patterns (21.5%) and enhancer-like chromatin signatures (60.9%). Macrophage activation, particularly to the M1 phenotype, markedly altered the lincRNA expression profiles, revealing 96 lincRNAs differentially expressed, suggesting potential roles in regulating macrophage inflammatory functions. A subset of lincRNAs overlapped genomewide association study loci for cardiometabolic disorders. MacORIS (macrophage-enriched obesity-associated lincRNA serving as a repressor of IFN-γ signaling), a macrophage-enriched lincRNA not expressed in mouse macrophages, harbors variants associated with central obesity. Knockdown of MacORIS, which is located in the cytoplasm, enhanced IFN-γ-induced JAK2 (Janus kinase 2) and STAT1 (signal transducer and activator of transcription 1) phosphorylation in THP-1 macrophages, suggesting a potential role as a repressor of IFN-γ signaling. Induced pluripotent stem cell-derived macrophages recapitulated the lincRNA transcriptome of human monocyte-derived macrophages and provided a high-fidelity model with which to study lincRNAs in human macrophage biology, particularly those not conserved in mouse. CONCLUSIONS: High-resolution transcriptomics identified lincRNAs that form part of the coordinated response during macrophage activation, including specific macrophage lincRNAs associated with human cardiometabolic disorders that modulate macrophage inflammatory functions.
Assuntos
Doenças Cardiovasculares/genética , Regulação da Expressão Gênica , Ativação de Macrófagos/genética , Macrófagos/metabolismo , Síndrome Metabólica/genética , RNA Longo não Codificante/genética , RNA/genética , Doenças Cardiovasculares/metabolismo , Doenças Cardiovasculares/patologia , Células Cultivadas , Estudo de Associação Genômica Ampla , Sequenciamento de Nucleotídeos em Larga Escala , Humanos , Macrófagos/patologia , Síndrome Metabólica/metabolismo , Síndrome Metabólica/patologia , RNA Longo não Codificante/biossíntese , Reação em Cadeia da Polimerase Via Transcriptase ReversaRESUMO
CONTEXT: Adipose inflammation is a crucial link between obesity and its metabolic complications. Human experimental endotoxemia is a controlled model for the study of inflammatory cardiometabolic responses in vivo. OBJECTIVE: We hypothesized that adipose genes down-regulated during endotoxemia would approximate changes observed with obesity-related inflammation and reveal novel candidates in cardiometabolic disease. DESIGN, SUBJECTS, AND INTERVENTION: Healthy volunteers (n = 14) underwent a 3 ng/kg endotoxin challenge; adipose biopsies were taken at 0, 4, 12, and 24 h for mRNA microarray. A priority list of highly down-regulated and biologically relevant genes was validated by RT-PCR in an independent sample of adipose from healthy subjects (n = 7) undergoing a subclinical 0.6 ng/kg endotoxemia protocol. Expression of validated genes was screened in adipose of lean and severely obese individuals (n = 11 per group), and cellular source was probed in cultured adipocytes and macrophages. RESULTS: Endotoxemia (3 ng/kg) suppressed expression of 353 genes (to <67% of baseline; P < 1 × 10(-5)) of which 68 candidates were prioritized for validation. In low-dose (0.6 ng/kg) endotoxin validation, 22 (32%) of these 68 genes were confirmed. Functional classification revealed that many of these genes are involved in cell development and differentiation. Of validated genes, 59% (13 of 22) were down-regulated more than 1.5-fold in primary human adipocytes after treatment with endotoxin. In human macrophages, 59% (13 of 22) were up-regulated during differentiation to inflammatory M1 macrophages whereas 64% (14 of 22) were down-regulated during transition to homeostatic M2 macrophages. Finally, in obese vs. lean adipose, 91% (20 of 22) tended to have reduced expression (χ(2) = 10.72, P < 0.01) with 50% (11 of 22) reaching P < 0.05 (χ(2) = 9.28, P < 0.01). CONCLUSIONS: Exploration of down-regulated mRNA in adipose during human endotoxemia revealed suppression of genes involved in cell development and differentiation. A majority of candidates were also suppressed in endogenous human obesity, suggesting a potential pathophysiological role in human obesity-related adipose inflammation.
Assuntos
Tecido Adiposo/metabolismo , Regulação para Baixo , Endotoxemia/genética , Obesidade/metabolismo , Adolescente , Adulto , Endotoxemia/induzido quimicamente , Endotoxemia/metabolismo , Feminino , Humanos , Inflamação/genética , Inflamação/metabolismo , Macrófagos/metabolismo , Masculino , Obesidade/genéticaRESUMO
OBJECTIVES: This study sought to examine the role of lipoprotein-associated phospholipase A2 (Lp-PLA2/PLA2G7) in human inflammation and coronary atherosclerosis. BACKGROUND: Lp-PLA2 has emerged as a potential therapeutic target in coronary heart disease. Data supporting Lp-PLA2 are indirect and confounded by species differences; whether Lp-PLA2 is causal in coronary heart disease remains in question. METHODS: We examined inflammatory regulation of Lp-PLA2 during experimental endotoxemia in humans, probed the source of Lp-PLA2 in human leukocytes under inflammatory conditions, and assessed the relationship of variation in PLA2G7, the gene encoding Lp-PLA2, with coronary artery calcification. RESULTS: In contrast to circulating tumor necrosis factor-alpha and C-reactive protein, blood and monocyte Lp-PLA2 messenger ribonucleic acid decreased transiently, and plasma Lp-PLA2 mass declined modestly during endotoxemia. In vitro, Lp-PLA2 expression increased dramatically during human monocyte to macrophage differentiation and further in inflammatory macrophages and foamlike cells. Despite only a marginal association of single nucleotide polymorphisms in PLA2G7 with Lp-PLA2 activity or mass, numerous PLA2G7 single nucleotide polymorphisms were associated with coronary artery calcification. In contrast, several single nucleotide polymorphisms in CRP were significantly associated with plasma C-reactive protein levels but had no relation with coronary artery calcification. CONCLUSIONS: Circulating Lp-PLA2 did not increase during acute phase response in humans, whereas inflammatory macrophages and foam cells, but not circulating monocytes, are major leukocyte sources of Lp-PLA2. Common genetic variation in PLA2G7 is associated with subclinical coronary atherosclerosis. These data link Lp-PLA2 to atherosclerosis in humans while highlighting the challenge in using circulating Lp-PLA2 as a biomarker of Lp-PLA2 actions in the vasculature.
Assuntos
1-Alquil-2-acetilglicerofosfocolina Esterase/genética , Doença da Artéria Coronariana/genética , Regulação da Expressão Gênica , Inflamação/genética , RNA Mensageiro/genética , 1-Alquil-2-acetilglicerofosfocolina Esterase/metabolismo , Adulto , Biomarcadores/metabolismo , Doença da Artéria Coronariana/enzimologia , Doença da Artéria Coronariana/patologia , Feminino , Humanos , Inflamação/enzimologia , Masculino , Polimorfismo de Nucleotídeo Único , Reação em Cadeia da Polimerase em Tempo RealRESUMO
OBJECTIVES: Inflammation may directly impair HDL functions, in particular reverse cholesterol transport (RCT), but limited data support this concept in humans. METHODS AND RESULTS: We employed low-dose human endotoxemia to assess the effects of inflammation on HDL and RCT-related parameters in vivo. Endotoxemia induced remodelling of HDL with depletion of pre-ß1a HDL particles determined by 2-D gel electrophoresis (-32.2±9.3% at 24 h, p<0.05) as well as small (-23.0±5.1%, p<0.01, at 24 h) and medium (-57.6±8.0% at 16 h, p<0.001) HDL estimated by nuclear magnetic resonance (NMR). This was associated with induction of class II secretory phospholipase A2 (~36 fold increase) and suppression of lecithin:cholesterol acyltransferase activity (-20.8±3.4% at 24 h, p<0.01) and cholesterol ester transfer protein mass (-22.2±6.8% at 24 h, p<0.001). The HDL fraction, isolated following endotoxemia, had reduced capacity to efflux cholesterol in vitro from SR-BI and ABCA1, but not ABCG1 transporter cell models. CONCLUSIONS: These data support the concept that "atherogenic-HDL dysfunction" and impaired RCT occur in human inflammatory syndromes, largely independent of changes in plasma HDL-C and ApoA-I levels.
Assuntos
HDL-Colesterol/sangue , Lipoproteínas de Alta Densidade Pré-beta/sangue , Inflamação/sangue , Transportador 1 de Cassete de Ligação de ATP , Membro 1 da Subfamília G de Transportadores de Cassetes de Ligação de ATP , Transportadores de Cassetes de Ligação de ATP/metabolismo , Adulto , Animais , Apolipoproteína A-I/sangue , Linhagem Celular , Proteínas de Transferência de Ésteres de Colesterol/metabolismo , Eletroforese em Gel Bidimensional , Endotoxemia/sangue , Endotoxemia/complicações , Feminino , Fosfolipases A2 do Grupo II/metabolismo , Humanos , Inflamação/etiologia , Espectroscopia de Ressonância Magnética , Masculino , Camundongos , Tamanho da Partícula , Fosfatidilcolina-Esterol O-Aciltransferase/metabolismo , Ratos , Receptores Depuradores Classe B/metabolismo , Fatores de Tempo , Adulto JovemRESUMO
OBJECTIVE: Leukocyte infiltration of adipose is a critical determinant of obesity-related metabolic diseases. Fractalkine (CX3CL1) and its receptor (CX3CR1) comprise a chemokine system involved in leukocyte recruitment and adhesion in atherosclerosis, but its role in adipose inflammation and type 2 diabetes is unknown. RESEARCH DESIGN AND METHODS: CX3CL1 mRNA and protein were quantified in subcutaneous adipose and blood during experimental human endotoxemia and in lean and obese human adipose. CX3CL1 cellular source was probed in human adipocytes, monocytes, and macrophages, and CX3CL1-blocking antibodies were used to assess its role in monocyte-adipocyte adhesion. The association of genetic variation in CX3CR1 with metabolic traits was determined in a community-based sample. Finally, plasma CX3CL1 levels were measured in a case-control study of type 2 diabetes. RESULTS: Endotoxemia induced adipose CX3CL1 mRNA (32.7-fold, P < 1 × 10(-5)) and protein (43-fold, P = 0.006). Obese subjects had higher CX3CL1 levels in subcutaneous adipose compared with lean (0.420 ± 0.387 vs. 0.228 ± 0.187 ng/mL, P = 0.04). CX3CL1 was expressed and secreted by human adipocytes and stromal vascular cells. Inflammatory cytokine induction of CX3CL1 in human adipocytes (27.5-fold mRNA and threefold protein) was completely attenuated by pretreatment with a peroxisome proliferator-activated receptor-γ agonist. A putative functional nonsynonymous single nucleotide polymorphism (rs3732378) in CX3CR1 was associated with adipose and metabolic traits, and plasma CX3CL1 levels were increased in patients with type 2 diabetes vs. nondiabetics (0.506 ± 0.262 vs. 0.422 ± 0.210 ng/mL, P < 0.0001). CONCLUSIONS: CX3CL1-CX3CR1 is a novel inflammatory adipose chemokine system that modulates monocyte adhesion to adipocytes and is associated with obesity, insulin resistance, and type 2 diabetes. These data provide support for CX3CL1 as a diagnostic and therapeutic target in cardiometabolic disease.
Assuntos
Quimiocina CX3CL1/sangue , Quimiocina CX3CL1/metabolismo , Diabetes Mellitus Tipo 2/sangue , Diabetes Mellitus Tipo 2/metabolismo , Adipócitos/citologia , Adipócitos/metabolismo , Adolescente , Adulto , Western Blotting , Receptor 1 de Quimiocina CX3C , Adesão Celular/genética , Adesão Celular/fisiologia , Linhagem Celular , Células Cultivadas , Quimiocina CX3CL1/genética , Diabetes Mellitus Tipo 2/genética , Ensaio de Imunoadsorção Enzimática , Feminino , Humanos , Inflamação/metabolismo , Leucócitos Mononucleares/citologia , Leucócitos Mononucleares/metabolismo , Masculino , Obesidade/genética , Obesidade/metabolismo , PPAR gama/agonistas , PPAR gama/metabolismo , Reação em Cadeia da Polimerase , Polimorfismo Genético/genética , Receptores de Quimiocinas/genética , Receptores de Quimiocinas/metabolismo , Adulto JovemRESUMO
OBJECTIVE: An emerging model of metabolic syndrome and type 2 diabetes is of adipose dysfunction with leukocyte recruitment into adipose leading to chronic inflammation and insulin resistance (IR). This study sought to explore potential mechanisms of inflammatory-induced IR in humans with a focus on adipose tissue. RESEARCH DESIGN AND METHODS: We performed a 60-h endotoxemia protocol (3 ng/kg intravenous bolus) in healthy adults (n = 20, 50% male, 80% Caucasian, aged 27.3 +/- 4.8 years). Before and after endotoxin, whole-blood sampling, subcutaneous adipose biopsies, and frequently sampled intravenous glucose tolerance (FSIGT) testing were performed. The primary outcome was the FSIGT insulin sensitivity index (S(i)). Secondary measures included inflammatory and metabolic markers and whole-blood and adipose mRNA and protein expression. RESULTS: Endotoxemia induced systemic IR as demonstrated by a 35% decrease in S(i) (3.17 +/- 1.66 to 2.06 +/- 0.73 x 10(-4) [microU * ml(-1) * min(-1)], P < 0.005), while there was no effect on pancreatic beta-cell function. In adipose, endotoxemia suppressed insulin receptor substrate-1 and markedly induced suppressor of cytokine signaling proteins (1 and 3) coincident with local activation of innate (interleukin-6, tumor necrosis factor) and adaptive (monocyte chemoattractant protein-1 and CXCL10 chemokines) inflammation. These changes are known to attenuate insulin receptor signaling in model systems. CONCLUSIONS: We demonstrate, for the first time in humans, that acute inflammation induces systemic IR following modulation of specific adipose inflammatory and insulin signaling pathways. It also provides a rationale for focused mechanistic studies and a model for human proof-of-concept trials of novel therapeutics targeting adipose inflammation in IR and related consequences in humans.
Assuntos
Endotoxemia/fisiopatologia , Inflamação/fisiopatologia , Resistência à Insulina/fisiologia , Absorciometria de Fóton , Doença Aguda , Tecido Adiposo/fisiopatologia , Adulto , Pressão Sanguínea , Proteínas Sanguíneas/genética , Quimiocinas/genética , HDL-Colesterol/sangue , Citocinas/genética , Diabetes Mellitus Tipo 2/genética , Diabetes Mellitus Tipo 2/fisiopatologia , Feminino , Teste de Tolerância a Glucose , Humanos , Inflamação/genética , Resistência à Insulina/genética , Lipopolissacarídeos/sangue , Masculino , RNA Mensageiro/genética , Proteínas Supressoras da Sinalização de Citocina/genética , Receptores Toll-Like/genética , Adulto JovemRESUMO
OBJECTIVE: Adipose inflammation plays a central role in obesity-related metabolic and cardiovascular complications. However, few human adipose-secreted proteins are known to mediate these processes. We hypothesized that microarray mRNA profiling of human adipose during evoked inflammation could identify novel adipocytokines. RESEARCH DESIGN AND METHODS: Healthy human volunteers (n = 14) were treated with intravenous endotoxin (3 ng/kg lipopolysaccharide [LPS]) and underwent subcutaneous adipose biopsies before and after LPS. On Affymetrix U133Plus 2.0 arrays, adipose mRNAs modulated >1.5-fold (with P < 0.00001) were selected. SignalP 3.0 and SecretomeP 2.0 identified genes predicted to encode secreted proteins. Of these, 86 candidates were chosen for validation in adipose from an independent human endotoxemia protocol (N = 7, with 0.6 ng/kg LPS) and for exploration of cellular origin in primary human adipocytes and macrophages in vitro. RESULTS: Microarray identified 776 adipose genes modulated by LPS; 298 were predicted to be secreted. Of detectable prioritized genes, 82 of 85 (96% [95% CI 90-99]) were upregulated (fold changes >1.0) during the lower-dose (LPS 0.6 ng/kg) validation study and 51 of 85 (59% [49-70]) were induced greater than 1.5-fold. Treatment of primary adipocytes with LPS and macrophage polarization to M1 proinflammatory phenotype increased expression by 1.5-fold for 58 and 73% of detectable genes, respectively. CONCLUSIONS: We demonstrate that evoked inflammation of human adipose in vivo modulated expression of multiple genes likely secreted by adipocytes and monocytes. These included established adipocytokines and chemokines implicated in recruitment and activation of lymphocytes, adhesion molecules, antioxidants, and several novel genes with unknown function. Such candidates may represent biomarkers and therapeutic targets for obesity-related complications.