RESUMO
Idiopathic pulmonary fibrosis (IPF) is the most severe form of chronic lung fibrosis. Circulating monocytes have been implicated in immune pathology in IPF but their phenotype is unknown. In this work, we determined the immune phenotype of monocytes in IPF using multi-colour flow cytometry, RNA sequencing and corresponding serum factors, and mapped the main findings to amount of lung fibrosis and single cell transcriptomic landscape of myeloid cells in IPF lungs. We show that monocytes from IPF patients displayed increased expression of CD64 (FcγR1) which correlated with amount of lung fibrosis, and an amplified type I IFN response ex vivo. These were accompanied by markedly raised CSF-1 levels, IL-6, and CCL-2 in serum of IPF patients. Interrogation of single cell transcriptomic data from human IPF lungs revealed increased proportion of CD64hi monocytes and "transitional macrophages" with higher expression of CCL-2 and type I IFN genes. Our study shows that monocytes in IPF patients are phenotypically distinct from age-matched controls, with a primed type I IFN pathway that may contribute to driving chronic inflammation and fibrosis. These findings strengthen the potential role of monocytes in the pathogenesis of IPF.
Assuntos
Fibrose Pulmonar Idiopática/imunologia , Interferon Tipo I/metabolismo , Pulmão/imunologia , Monócitos/imunologia , Estudos de Casos e Controles , Células Cultivadas , Quimiocina CCL2/sangue , Citometria de Fluxo , Perfilação da Expressão Gênica , Humanos , Fibrose Pulmonar Idiopática/genética , Fibrose Pulmonar Idiopática/metabolismo , Fibrose Pulmonar Idiopática/patologia , Imunofenotipagem , Interferon Tipo I/genética , Interleucina-6/sangue , Pulmão/metabolismo , Pulmão/patologia , Fator Estimulador de Colônias de Macrófagos/sangue , Macrófagos/imunologia , Macrófagos/metabolismo , Monócitos/metabolismo , Fenótipo , Receptores de IgG/genética , Receptores de IgG/metabolismo , Análise de Célula ÚnicaRESUMO
Diseases of accelerated aging often occur together (multimorbidity), and their prevalence is increasing, with high societal and health care costs. Chronic obstructive pulmonary disease (COPD) is one such condition, in which one half of patients exhibit ≥4 age-related diseases. Diseases of accelerated aging share common molecular pathways, which lead to the detrimental accumulation of senescent cells. These senescent cells no longer divide but release multiple inflammatory proteins, known as the senescence-associated secretory phenotype, which may perpetuate and speed disease. Here, we show that inhibiting miR-570-3p, which is increased in COPD cells, reverses cellular senescence by restoring the antiaging molecule sirtuin-1. MiR-570-3p is induced by oxidative stress in airway epithelial cells through p38 MAP kinase-c-Jun signaling and drives senescence by inhibiting sirtuin-1. Inhibition of elevated miR-570-3p in COPD small airway epithelial cells, using an antagomir, restores sirtuin-1 and suppresses markers of cellular senescence (p16INK4a, p21Waf1, and p27Kip1), thereby restoring cellular growth by allowing progression through the cell cycle. MiR-570-3p inhibition also suppresses the senescence-associated secretory phenotype (matrix metalloproteinases-2/9, C-X-C motif chemokine ligand 8, IL-1ß, and IL-6). Collectively, these data suggest that inhibiting miR-570-3p rejuvenates cells via restoration of sirtuin-1, reducing many of the abnormalities associated with cellular senescence.-Baker, J. R., Vuppusetty, C., Colley, T., Hassibi, S., Fenwick, P. S., Donnelly, L. E., Ito, K., Barnes, P. J. MicroRNA-570 is a novel regulator of cellular senescence and inflammaging.
Assuntos
Senescência Celular/fisiologia , Inflamação/fisiopatologia , MicroRNAs/fisiologia , Idoso , Linhagem Celular , Feminino , Humanos , Masculino , MicroRNAs/metabolismo , Pessoa de Meia-Idade , Estresse Oxidativo , Ligação Proteica , Proteínas Proto-Oncogênicas c-jun/metabolismo , Doença Pulmonar Obstrutiva Crônica/fisiopatologia , Transdução de Sinais , Sirtuína 1/metabolismo , Proteínas Quinases p38 Ativadas por Mitógeno/metabolismoRESUMO
SIRT1 (silent information regulator 2 homolog 1) is a crucial cellular survival protein especially in oxidative stress environments, and has been thought to locate within the nuclei, but also known to shuttle between cytoplasm and nuclei in some cell types. Here, we show for the first time the dynamics of SIRT1 in the presence of single or concurrent cigarette smoke extract (CSE) exposure in human bronchial epithelial cells (HBEC). In BEAS-2B HBEC or primary HBEC, SIRT1 was localized predominantly in cytoplasm, and the CSE (3%) induced nuclear translocation of SIRT1 from cytoplasm in the presence of L-buthionine sulfoximine (an irreversible inhibitor of γ-glutamylcystein synthetase), mainly through the activation of phosphatidylinositol 3-kinase (PI3K) α subunit. This SIRT1 nuclear shuttling was associated with FOXO3a nuclear translocation and the strong induction of several anti-oxidant genes including superoxide dismutase (SOD) 2 and 3; therefore seemed to be an adaptive response. When BEAS-2B cells were pretreated with repeated exposure to a lower concentration of CSE (0.3%), the CSE-induced SIRT1 shuttling and resultant SOD2/3 mRNA induction were significantly impaired. Thus, this result offers a useful cell model to mimic the impaired anti-oxidant capacity in cigarette smoking-associated lung disease such as chronic obstructive pulmonary disease.
Assuntos
Brônquios/citologia , Células Epiteliais/efeitos dos fármacos , Sirtuína 1/metabolismo , Fumar/efeitos adversos , Western Blotting , Brônquios/efeitos dos fármacos , Brônquios/metabolismo , Núcleo Celular/metabolismo , Citoplasma/metabolismo , Relação Dose-Resposta a Droga , Células Epiteliais/metabolismo , Humanos , Estresse Oxidativo/efeitos dos fármacos , Reação em Cadeia da Polimerase Via Transcriptase ReversaRESUMO
Corticosteroid resistance is a major barrier to the effective treatment of chronic obstructive pulmonary disease (COPD). Oxidative stress from cigarette smoke and chronic inflammation is likely to induce this corticosteroid insensitivity. Quercetin is a polyphenol that has been reported to be an active oxygen scavenger as well as a functional adenosine monophosphate-activated protein kinase (AMPK) activator. The aim of this study was to investigate the effect of quercetin on corticosteroid responsiveness in COPD cells. Corticosteroid sensitivity was examined in human monocytic U937 cells exposed to cigarette smoke extract (CSE) and peripheral blood mononuclear cells (PBMC) collected from patients with COPD. Corticosteroid sensitivity was determined as the dexamethasone concentration causing 40% inhibition of tumor necrosis factor alpha-induced CXCL8 production (Dex-IC40) in the presence or absence of quercetin. In U937 cells, treatment with quercetin activated AMPK and induced expression of nuclear factor erythroid 2-related factor 2, and consequently reversed CSE-induced corticosteroid insensitivity. PBMC from patients with COPD showed corticosteroid insensitivity compared with those from healthy volunteers, and treatment with quercetin restored corticosteroid sensitivity. In conclusion, quercetin restores corticosteroid sensitivity, and has the potential to be a novel treatment in combination with corticosteroids in COPD.
Assuntos
Corticosteroides/farmacologia , Doença Pulmonar Obstrutiva Crônica/tratamento farmacológico , Quercetina/farmacologia , Adulto , Idoso , Estudos de Casos e Controles , Feminino , Humanos , Leucócitos Mononucleares/efeitos dos fármacos , Leucócitos Mononucleares/patologia , Masculino , Pessoa de Meia-Idade , Doença Pulmonar Obstrutiva Crônica/patologia , Quercetina/uso terapêutico , Fumaça/efeitos adversos , Produtos do Tabaco/efeitos adversos , Células Tumorais Cultivadas , Células U937RESUMO
The phosphatidylinositol 3-kinase (PI3K) pathway is activated in chronic obstructive pulmonary disease (COPD), but the regulatory mechanisms for this pathway are yet to be elucidated. The aim of this study was to determine the expression and role of phosphatase and tensin homolog deleted from chromosome 10 (PTEN), a negative regulator of the PI3K pathway, in COPD. PTEN protein expression was measured in the peripheral lung of COPD patients compared with smoking and nonsmoking controls. The direct influence of cigarette smoke extract (CSE) on PTEN expression was assessed using primary lung epithelial cells and a cell line (BEAS-2B) in the presence or absence of l-buthionine-sulfoximine (BSO) to deplete intracellular glutathione. The impact of PTEN knockdown by RNA interference on cytokine production was also examined. In peripheral lung, PTEN protein was significantly decreased in patients with COPD compared with the subjects without COPD (P < 0.001) and positively correlated with the severity of airflow obstruction (forced expiratory volume in 1-s percent predicted; r = 0.50; P = 0.0012). Conversely, phosphorylated Akt, as a marker of PI3K activation, showed a negative correlation with PTEN protein levels (r = -0.41; P = 0.0042). In both primary bronchial epithelial cells and BEAS-2B cells, CSE decreased PTEN protein, which was reversed by N-acetyl cysteine treatment. PTEN knockdown potentiated Akt phosphorylation and enhanced production of proinflammatory cytokines, such as IL-6, CXCL8, CCL2, and CCL5. In conclusion, oxidative stress reduces PTEN protein levels, which may result in increased PI3K signaling and amplification of inflammation in COPD.
Assuntos
Citocinas/metabolismo , Inflamação/metabolismo , PTEN Fosfo-Hidrolase/metabolismo , Fosfatidilinositol 3-Quinases/metabolismo , Doença Pulmonar Obstrutiva Crônica/metabolismo , Idoso , Linhagem Celular , Células Epiteliais/metabolismo , Feminino , Humanos , Pulmão/metabolismo , Masculino , Estresse Oxidativo/fisiologia , Fosforilação/fisiologia , Proteínas Proto-Oncogênicas c-akt/metabolismo , Transdução de Sinais/fisiologia , Fumaça/efeitos adversos , Fumar/efeitos adversosRESUMO
RATIONALE: Corticosteroid resistance is a major barrier to the effective treatment of chronic obstructive pulmonary disease (COPD). Several molecular mechanisms have been proposed, such as activations of the phosphoinositide-3-kinase/Akt pathway and p38 mitogen-activated protein kinase. However, the mechanism for corticosteroid resistance is still not fully elucidated. OBJECTIVES: To investigate the role of mammalian target of rapamycin (mTOR) in corticosteroid sensitivity in COPD. METHODS: The corticosteroid sensitivity of peripheral blood mononuclear cells collected from patients with COPD, smokers, and nonsmoking control subjects, or of human monocytic U937 cells exposed to cigarette smoke extract (CSE), was quantified as the dexamethasone concentration required to achieve 30% inhibition of tumor necrosis factor-α-induced CXCL8 production in the presence or absence of the mTOR inhibitor rapamycin. mTOR activity was determined as the phosphorylation of p70 S6 kinase, using Western blotting. MEASUREMENTS AND MAIN RESULTS: mTOR activity was increased in peripheral blood mononuclear cells from patients with COPD, and treatment with rapamycin inhibited this as well as restoring corticosteroid sensitivity. In U937 cells, CSE stimulated mTOR activity and c-Jun expression, but pretreatment with rapamycin inhibited both and also reversed CSE-induced corticosteroid insensitivity. CONCLUSIONS: mTOR inhibition by rapamycin restores corticosteroid sensitivity via inhibition of c-Jun expression, and thus mTOR is a potential novel therapeutic target for COPD.
Assuntos
Corticosteroides/farmacologia , Resistência a Medicamentos/efeitos dos fármacos , Estresse Oxidativo/efeitos dos fármacos , Proteínas Proto-Oncogênicas c-jun/efeitos dos fármacos , Doença Pulmonar Obstrutiva Crônica/tratamento farmacológico , Sirolimo/farmacologia , Serina-Treonina Quinases TOR/efeitos dos fármacos , Corticosteroides/uso terapêutico , Idoso , Resistência a Medicamentos/imunologia , Feminino , Histona Desacetilase 2/efeitos dos fármacos , Histona Desacetilase 2/fisiologia , Humanos , Imunossupressores/imunologia , Imunossupressores/farmacologia , Imunossupressores/uso terapêutico , Masculino , Pessoa de Meia-Idade , Estresse Oxidativo/fisiologia , Fosfatidilinositol 3-Quinases/efeitos dos fármacos , Fosfatidilinositol 3-Quinases/fisiologia , Proteínas Proto-Oncogênicas c-akt/efeitos dos fármacos , Proteínas Proto-Oncogênicas c-akt/fisiologia , Proteínas Proto-Oncogênicas c-jun/fisiologia , Doença Pulmonar Obstrutiva Crônica/imunologia , Doença Pulmonar Obstrutiva Crônica/fisiopatologia , Sirolimo/imunologia , Sirolimo/uso terapêutico , Fumar/efeitos adversos , Fumar/fisiopatologia , Serina-Treonina Quinases TOR/fisiologia , Células U937/efeitos dos fármacos , Proteínas Quinases p38 Ativadas por Mitógeno/efeitos dos fármacos , Proteínas Quinases p38 Ativadas por Mitógeno/fisiologiaRESUMO
Histone deacetylases (HDACs) are key molecules involved in epigenetic regulation of gene expression. We have previously demonstrated that oxidative stress caused a reduction in HDAC2, resulting in amplified inflammation and reduced corticosteroid responsiveness. Here we showed nitrative/oxidative stress reduced HDAC2 expression via nitration of distinct tyrosine residues. Peroxynitrite, hydrogen peroxide and cigarette smoke-conditioned medium reduced HDAC2 expression in A549 epithelial cells in vitro. This reduction was due to increased proteasomal degradation following ubiquitination rather than reduction of mRNA expression or stability. HDAC2 was nitrated under nitrative/oxidative stress and in the peripheral lung tissues of smokers and patients with chronic obstructive pulmonary disease. Mutagenesis studies replacing tyrosine (Y) residues with alanine revealed that Y253 is at least partly responsible for the proteasomal degradation of HDAC2 under nitrative stress. Thus, nitration of distinct tyrosine residues modifies both the expression and activity of HDAC2, having an impact on epigenetic regulation.
Assuntos
Epigênese Genética , Histona Desacetilases/metabolismo , Nitratos/metabolismo , Proteínas Repressoras/metabolismo , Tirosina/metabolismo , Linhagem Celular , Histona Desacetilase 2 , Histona Desacetilases/genética , Humanos , Estresse Oxidativo , Complexo de Endopeptidases do Proteassoma/metabolismo , Proteínas Repressoras/genética , Tirosina/genéticaRESUMO
Inappropriate elevation of matrix metalloproteinase-9 (MMP9) is reported to be involved in the pathogenesis of chronic obstructive pulmonary disease (COPD). The object of this study was to identify the molecular mechanism underlying this increase of MMP9 expression, and here we show that oxidative stress-dependent reduction of a protein deacetylase, SIRT1, known as a putative antiaging enzyme, causes elevation of MMP9 expression. A sirtuin inhibitor, splitomycin, and SIRT1 knockdown by RNA interference led an increase in MMP9 expression in human monocytic U937 cells and in primary sputum macrophages, which was detected by RT-PCR, Western blot, activity assay, and zymography. In fact, the SIRT1 level was significantly decreased in peripheral lungs of patients with COPD, and this increase was inversely correlated with MMP9 expression and MMP9 promoter activation detected by a chromatin immunoprecipitation assay. H(2)O(2) reduced SIRT1 expression and activity in U937 cells; furthermore, cigarette smoke exposure also caused reduction of SIRT1 expression in lung tissue of A/J mice, with concomitant elevation of MMP9. Intranasal treatment of a selective and novel SIRT1 small molecule activator, SRT2172, blocked the increase of MMP9 expression in the lung as well as pulmonary neutrophilia and the reduction in exercise tolerance. Thus, SIRT1 is a negative regulator of MMP9 expression, and SIRT1 activation is implicated as a novel therapeutic approach to treating chronic inflammatory diseases, in which MMP9 is abundant.