RESUMO
OBJECTIVE: Human macrophages can shift phenotype across the inflammatory M1 and reparative M2 spectrum in response to environmental challenges, but the mechanisms promoting inflammatory and cardiometabolic disease-associated M1 phenotypes remain incompletely understood. Alternative splicing (AS) is emerging as an important regulator of cellular function, yet its role in macrophage activation is largely unknown. We investigated the extent to which AS occurs in M1 activation within the cardiometabolic disease context and validated a functional genomic cell model for studying human macrophage-related AS events. APPROACH AND RESULTS: From deep RNA-sequencing of resting, M1, and M2 primary human monocyte-derived macrophages, we found 3860 differentially expressed genes in M1 activation and detected 233 M1-induced AS events; the majority of AS events were cell- and M1-specific with enrichment for pathways relevant to macrophage inflammation. Using genetic variant data for 10 cardiometabolic traits, we identified 28 trait-associated variants within the genomic loci of 21 alternatively spliced genes and 15 variants within 7 differentially expressed regulatory splicing factors in M1 activation. Knockdown of 1 such splicing factor, CELF1, in primary human macrophages led to increased inflammatory response to M1 stimulation, demonstrating CELF1's potential modulation of the M1 phenotype. Finally, we demonstrated that an induced pluripotent stem cell-derived macrophage system recapitulates M1-associated AS events and provides a high-fidelity macrophage AS model. CONCLUSIONS: AS plays a role in defining macrophage phenotype in a cell- and stimulus-specific fashion. Alternatively spliced genes and splicing factors with trait-associated variants may reveal novel pathways and targets in cardiometabolic diseases.
Assuntos
Processamento Alternativo , Diferenciação Celular , Perfilação da Expressão Gênica , Células-Tronco Pluripotentes Induzidas/metabolismo , Inflamação/genética , Ativação de Macrófagos/genética , Macrófagos/metabolismo , Transcriptoma , Proteínas CELF1/genética , Proteínas CELF1/metabolismo , Células Cultivadas , Perfilação da Expressão Gênica/métodos , Redes Reguladoras de Genes , Estudo de Associação Genômica Ampla , Humanos , Inflamação/metabolismo , Lipopolissacarídeos/farmacologia , Ativação de Macrófagos/efeitos dos fármacos , Macrófagos/efeitos dos fármacos , Análise de Sequência com Séries de Oligonucleotídeos , Fenótipo , Mapeamento de Interação de Proteínas , Interferência de RNA , Transdução de Sinais , TransfecçãoRESUMO
Studies in human populations have shown a significant correlation between procollagen C-endopeptidase enhancer protein 2 (PCPE2) single nucleotide polymorphisms and plasma HDL cholesterol concentrations. PCPE2, a 52-kDa glycoprotein located in the extracellular matrix, enhances the cleavage of C-terminal procollagen by bone morphogenetic protein 1 (BMP1). Our studies here focused on investigating the basis for the elevated concentration of enlarged plasma HDL in PCPE2-deficient mice to determine whether they protected against diet-induced atherosclerosis. PCPE2-deficient mice were crossed with LDL receptor-deficient mice to obtain LDLr(-/-), PCPE2(-/-) mice, which had elevated HDL levels compared with LDLr(-/-) mice with similar LDL concentrations. We found that LDLr(-/-), PCPE2(-/-) mice had significantly more neutral lipid and CD68+ infiltration in the aortic root than LDLr(-/-) mice. Surprisingly, in light of their elevated HDL levels, the extent of aortic lipid deposition in LDLr(-/-), PCPE2(-/-) mice was similar to that reported for LDLr(-/-), apoA-I(-/-) mice, which lack any apoA-I/HDL. Furthermore, LDLr(-/-), PCPE2(-/-) mice had reduced HDL apoA-I fractional clearance and macrophage to fecal reverse cholesterol transport rates compared with LDLr(-/-) mice, despite a 2-fold increase in liver SR-BI expression. PCPE2 was shown to enhance SR-BI function by increasing the rate of HDL-associated cholesteryl ester uptake, possibly by optimizing SR-BI localization and/or conformation. We conclude that PCPE2 is atheroprotective and an important component of the reverse cholesterol transport HDL system.
Assuntos
Aterosclerose/metabolismo , Aterosclerose/prevenção & controle , Ésteres do Colesterol/metabolismo , Glicoproteínas/metabolismo , Lipoproteínas HDL/metabolismo , Receptores Depuradores Classe B/metabolismo , Animais , Antígenos CD/genética , Antígenos CD/metabolismo , Antígenos de Diferenciação Mielomonocítica/genética , Antígenos de Diferenciação Mielomonocítica/metabolismo , Aorta/metabolismo , Aorta/patologia , Apolipoproteína A-I/genética , Apolipoproteína A-I/metabolismo , Aterosclerose/induzido quimicamente , Aterosclerose/genética , Aterosclerose/patologia , Transporte Biológico Ativo/genética , Células CHO , Ésteres do Colesterol/genética , Cricetulus , Glicoproteínas/genética , Humanos , Peptídeos e Proteínas de Sinalização Intracelular , Lipoproteínas HDL/genética , Macrófagos/metabolismo , Macrófagos/patologia , Camundongos , Camundongos Knockout , Receptores de LDL/genética , Receptores de LDL/metabolismo , Receptores Depuradores Classe B/genéticaRESUMO
Relapse rates in high-risk neuroblastoma remain exceedingly high. The malignant cells that are responsible for relapse have not been identified, and mechanisms of therapy resistance remain poorly understood. Here, we used single nucleus RNA sequencing and bulk whole genome sequencing to identify and characterize the residual malignant persister cells that survive chemotherapy from a cohort of 20 matched diagnosis and definitive surgery tumor samples from patients treated with high-risk neuroblastoma induction chemotherapy. We show that persister cells share common mechanisms of chemotherapy escape including suppression of MYCN activity and activation of NF-κB signaling, the latter is further enhanced by cell-cell communication between the malignant cells and the tumor microenvironment. Overall, our work dissects the transcriptional landscape of cellular persistence in high-risk neuroblastoma and paves the way to the development of new therapeutic strategies to prevent disease relapse.
RESUMO
Activating point mutations in Anaplastic Lymphoma Kinase (ALK) have positioned ALK as the only mutated oncogene tractable for targeted therapy in neuroblastoma. Cells with these mutations respond to lorlatinib in pre-clinical studies, providing the rationale for a first-in-child Phase 1 trial (NCT03107988) in patients with ALK-driven neuroblastoma. To track evolutionary dynamics and heterogeneity of tumors, and to detect early emergence of lorlatinib resistance, we collected serial circulating tumor DNA samples from patients enrolled on this trial. Here we report the discovery of off-target resistance mutations in 11 patients (27%), predominantly in the RAS-MAPK pathway. We also identify newly acquired secondary compound ALK mutations in 6 (15%) patients, all acquired at disease progression. Functional cellular and biochemical assays and computational studies elucidate lorlatinib resistance mechanisms. Our results establish the clinical utility of serial circulating tumor DNA sampling to track response and progression and to discover acquired resistance mechanisms that can be leveraged to develop therapeutic strategies to overcome lorlatinib resistance.
Assuntos
Carcinoma Pulmonar de Células não Pequenas , DNA Tumoral Circulante , Neoplasias Pulmonares , Neuroblastoma , Humanos , Aminopiridinas/uso terapêutico , Quinase do Linfoma Anaplásico/genética , Carcinoma Pulmonar de Células não Pequenas/genética , DNA Tumoral Circulante/genética , Resistencia a Medicamentos Antineoplásicos/genética , Lactamas Macrocíclicas/uso terapêutico , Neoplasias Pulmonares/genética , Mutação , Neuroblastoma/tratamento farmacológico , Neuroblastoma/genética , Inibidores de Proteínas Quinases/uso terapêuticoRESUMO
This report details the lipid composition of nascent HDL (nHDL) particles formed by the action of the ATP binding cassette transporter A1 (ABCA1) on apolipoprotein A-I (apoA-I). nHDL particles of different size (average diameters of â¼ 12, 10, 7.5, and <6 nm) and composition were purified by size-exclusion chromatography. Electron microscopy suggested that the nHDL were mostly spheroidal. The proportions of the principal nHDL lipids, free cholesterol, glycerophosphocholine, and sphingomyelin were similar to that of lipid rafts, suggesting that the lipid originated from a raft-like region of the cell. Smaller amounts of glucosylceramides, cholesteryl esters, and other glycerophospholipid classes were also present. The largest particles, â¼ 12 nm and 10 nm diameter, contained â¼ 43% free cholesterol, 2-3% cholesteryl ester, and three apoA-I molecules. Using chemical cross-linking chemistry combined with mass spectrometry, we found that three molecules of apoA-I in the â¼ 9-14 nm nHDL adopted a belt-like conformation. The smaller (7.5 nm diameter) spheroidal nHDL particles carried 30% free cholesterol and two molecules of apoA-I in a twisted, antiparallel, double-belt conformation. Overall, these new data offer fresh insights into the biogenesis and structural constraints involved in forming nascent HDL from ABCA1.
Assuntos
Transportadores de Cassetes de Ligação de ATP/metabolismo , Apolipoproteína A-I/química , Apolipoproteína A-I/metabolismo , Lipoproteínas HDL/biossíntese , Lipoproteínas HDL/química , Microdomínios da Membrana/metabolismo , Transportador 1 de Cassete de Ligação de ATP , Transporte Biológico , Ceramidas/metabolismo , Colesterol/metabolismo , Ácidos Graxos/metabolismo , Células HEK293 , Humanos , Lipoproteínas HDL/isolamento & purificação , Conformação Proteica , Esfingomielinas/metabolismoRESUMO
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: Atherosclerosis is a chronic inflammatory disorder whose development is inversely correlated with high-density lipoprotein concentration. Current therapies involve pharmaceuticals that significantly elevate plasma high-density lipoprotein cholesterol concentrations. Our studies were conducted to investigate the effects of low-dose lipid-free apolipoprotein A-I (apoA-I) on chronic inflammation. The aims of these studies were to determine how subcutaneously injected lipid-free apoA-I reduces accumulation of lipid and immune cells within the aortic root of hypercholesterolemic mice without sustained elevations in plasma high-density lipoprotein cholesterol concentrations. METHODS AND RESULTS: Ldlr-/- and Ldlr-/- apoA-I-/- mice were fed a Western diet for a total of 12 weeks. After 6 weeks, a subset of mice from each group received subcutaneous injections of 200 µg of lipid-free human apoA-I 3 times a week, while the other subset received 200 µg of albumin, as a control. Mice treated with lipid-free apoA-I showed a decrease in cholesterol deposition and immune cell retention in the aortic root compared with albumin-treated mice, regardless of genotype. This reduction in atherosclerosis appeared to be directly related to a decrease in the number of CD131 expressing cells and the esterified cholesterol to total cholesterol content in several immune cell compartments. In addition, apoA-I treatment altered microdomain cholesterol composition that shifted CD131, the common ß subunit of the interleukin 3 receptor, from lipid raft to nonraft fractions of the plasma membrane. CONCLUSIONS: ApoA-I treatment reduced lipid and immune cell accumulation within the aortic root by systemically reducing microdomain cholesterol content in immune cells. These data suggest that lipid-free apoA-I mediates beneficial effects through attenuation of immune cell lipid raft cholesterol content, which affects numerous types of signal transduction pathways that rely on microdomain integrity for assembly and activation.