RESUMO
Background: Hypersensitivity pneumonitis (HP) is an inflammatory disorder affecting lung parenchyma and often evolves into fibrosis (fHP). The altered regulation of genes involved in the pathogenesis of the disease is not well comprehended, while the role of microRNAs in lung fibroblasts remains unexplored. Methods: We used integrated bulk RNA-Seq and enrichment pathway bioinformatic analyses to identify differentially expressed (DE)-miRNAs and genes (DEGs) associated with HP lungs. In vitro, we evaluated the expression and potential role of miR-155-5p in the phenotype of fHP lung fibroblasts. Loss and gain assays were used to demonstrate the impact of miR-155-5p on fibroblast functions. In addition, mir-155-5p and its target TP53INP1 were analyzed after treatment with TGF-ß, IL-4, and IL-17A. Results: We found around 50 DEGs shared by several databases that differentiate HP from control and IPF lungs, constituting a unique HP lung transcriptional signature. Additionally, we reveal 18 DE-miRNAs that may regulate these DEGs. Among the candidates likely associated with HP pathogenesis was miR-155-5p. Our findings indicate that increased miR-155-5p in fHP fibroblasts coincides with reduced TP53INP1 expression, high proliferative capacity, and a lack of senescence markers compared to IPF fibroblasts. Induced overexpression of miR-155-5p in normal fibroblasts remarkably increases the proliferation rate and decreases TP53INP1 expression. Conversely, miR-155-5p inhibition reduces proliferation and increases senescence markers. TGF-ß, IL-4, and IL-17A stimulated miR-155-5p overexpression in HP lung fibroblasts. Conclusion: Our findings suggest a distinctive signature of 53 DEGs in HP, including CLDN18, EEF2, CXCL9, PLA2G2D, and ZNF683, as potential targets for future studies. Likewise, 18 miRNAs, including miR-155-5p, could be helpful to establish differences between these two pathologies. The overexpression of miR-155-5p and downregulation of TP53INP1 in fHP lung fibroblasts may be involved in his proliferative and profibrotic phenotype. These findings may help differentiate and characterize their pathogenic features and understand their role in the disease.
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Idiopathic pulmonary fibrosis (IPF) is an aging-associated disease characterized by exacerbated extracellular matrix deposition that disrupts oxygen exchange. Hypoxia and its transcription factors (HIF-1α and 2α) influence numerous circuits that could perpetuate fibrosis by increasing myofibroblasts differentiation and by promoting extracellular matrix accumulation. Therefore, this work aimed to elucidate the signature of hypoxia in the transcriptomic circuitry of IPF-derived fibroblasts. To determine this transcriptomic signature, a gene expression analysis with six lines of lung fibroblasts under normoxia or hypoxia was performed: three cell lines were derived from patients with IPF, and three were from healthy donors, a total of 36 replicates. We used the Clariom D platform, which allows us to evaluate a huge number of transcripts, to analyze the response to hypoxia in both controls and IPF. The control's response is greater by the number of genes and complexity. In the search for specific genes responsible for the IPF fibroblast phenotype, nineteen dysregulated genes were found in lung fibroblasts from IPF patients in hypoxia (nine upregulated and ten downregulated). In this sense, the signaling pathways revealed to be affected in the pulmonary fibroblasts of patients with IPF may represent an adaptation to chronic hypoxia.
Assuntos
Fibrose Pulmonar Idiopática , Fibroblastos/metabolismo , Humanos , Hipóxia/genética , Hipóxia/metabolismo , Fibrose Pulmonar Idiopática/genética , Fibrose Pulmonar Idiopática/metabolismo , Pulmão/metabolismo , Oxigênio/metabolismo , Fatores de Transcrição/metabolismo , Transcriptoma/genéticaRESUMO
Wood smoke (WS) contains many harmful compounds, including polycyclic aromatic hydrocarbons (PAHs). WS induces inflammation in the airways and lungs and can lead to the development of various acute and chronic respiratory diseases. Pulmonary fibroblasts are the main cells involved in the remodeling of the extracellular matrix (ECM) during the WS-induced inflammatory response. Although fibroblasts remain in a low proliferation state under physiological conditions, they actively participate in ECM remodeling during the inflammatory response in pathophysiological states. Consequently, we used normal human lung fibroblasts (NHLFs) to assess the potential effects of the PAHs-containing wood smoke extract (WSE) on the growth rate, total collagen synthesis, and the expression levels of collagen I and III, matrix metalloproteinase (MMP)-1, MMP-2, MMP-9, tissue inhibitor of metalloproteinase (TIMP)-1, TIMP-2, and the transforming growth factor (TGF)-ß1. We also assessed MMPs activity. The results showed that WSE induced a trimodal behavior in the growth rate curves in NHLFs; the growth rate increased with 0.5-1 % WSE and decreased with 2.5% WSE, without causing cell damage; 5-20% WSE inhibited the growth and induced cell damage. After 3 hours of exposure, 2.5% WSE induced an increase in total collagen synthesis and upregulation of TGF-ß1, collagen I and III, MMP-1, TIMP-1, and TIMP-2 expression. However, MMP-2 expression was downregulated and MMP-9 was not expressed. The gelatinase activity of MMP-2 was also increased. These results suggest that WSE affects the ECM remodeling in NHLFs and indicate the potential involvement of PAHs in this process.
Assuntos
Matriz Extracelular/efeitos dos fármacos , Fibroblastos/efeitos dos fármacos , Inflamação/induzido quimicamente , Inflamação/fisiopatologia , Pneumopatias/induzido quimicamente , Extratos Vegetais/efeitos adversos , Fumaça/efeitos adversos , Proliferação de Células/efeitos dos fármacos , Humanos , Magnoliopsida/química , Madeira/químicaRESUMO
Tuberculosis (TB), caused by Mycobacterium tuberculosis (Mtb), remains as a leading infectious cause of death worldwide. The increasing number of multidrug-resistant TB (MDR-TB) cases contributes to the poor control of the TB epidemic. Currently, little is known about the immunological requirements of protective responses against MDR-TB. This is of major relevance to identify immune markers for treatment monitoring and targets for adjuvant immunotherapies. Here, we hypothesized that MDR-TB patients display unique immunophenotypical features and immune cell migration dynamics compared to drug-sensitive TB (DS-TB). Hence, we prospectively conducted an extensive characterization of the immune profile of MDR-TB patients at different time points before and after pharmacological therapy. For this purpose, we focused on the leukocyte expression of chemokine receptors, distribution of different monocyte and lymphocyte subsets, plasma levels of chemotactic factors, and in vitro migration capacity of immune cells. Our comparative cohort consisted of DS-TB patients and healthy volunteer donors (HD). Our results demonstrate some unique features of leukocyte migration dynamics during MDR-TB. These include increased and prolonged circulation of CD3+ monocytes, CCR4+ monocytes, EM CD4+ T cells, EM/CM CD8+ T cells, and CXCR1+CXCR3+ T cells that is sustained even after the administration of anti-TB drugs. We also observed shared characteristics of both MDR-TB and DS-TB that include CCR2+ monocyte depletion in the blood; high plasma levels of MPC-1, CCL-7, and IP-10; and increased responsiveness of leukocytes to chemotactic signals in vitro. Our study contributes to a better understanding of the MDR-TB pathobiology and uncovers immunological readouts of treatment efficacy.
Assuntos
Antituberculosos/farmacologia , Leucócitos Mononucleares/imunologia , Receptores de Quimiocinas/metabolismo , Tuberculose Resistente a Múltiplos Medicamentos/imunologia , Tuberculose Pulmonar/imunologia , Adulto , Antituberculosos/uso terapêutico , Biomarcadores/análise , Biomarcadores/metabolismo , Estudos de Casos e Controles , Movimento Celular/imunologia , Monitoramento de Medicamentos/métodos , Seguimentos , Voluntários Saudáveis , Humanos , Leucócitos Mononucleares/metabolismo , Masculino , Testes de Sensibilidade Microbiana , Pessoa de Meia-Idade , Mycobacterium tuberculosis/efeitos dos fármacos , Mycobacterium tuberculosis/imunologia , Mycobacterium tuberculosis/isolamento & purificação , Estudos Prospectivos , Receptores de Quimiocinas/análise , Tuberculose Resistente a Múltiplos Medicamentos/sangue , Tuberculose Resistente a Múltiplos Medicamentos/tratamento farmacológico , Tuberculose Pulmonar/sangue , Tuberculose Pulmonar/tratamento farmacológico , Tuberculose Pulmonar/microbiologiaRESUMO
BACKGROUND: Around 8-10% of individuals over 50â years of age present interstitial lung abnormalities (ILAs), but their risk factors are uncertain. METHODS: From 817 individuals recruited in our lung ageing programme at the Mexican National Institute of Respiratory Diseases, 80 (9.7%) showed ILAs and were compared with 564 individuals of the same cohort with normal high-resolution computed tomography to evaluate demographic and functional differences, and with 80 individuals randomly selected from the same cohort for biomarkers. We evaluated MUC5B variant rs35705950, telomere length, and serum levels of matrix metalloproteinase (MMP)-1, MMP-2, MMP-3, MMP-7, MMP-8, MMP-9, MMP-12, MMP-13, interleukin (IL)-6, surfactant protein (SP)-D, α-Klotho and resistin. RESULTS: Individuals with ILAs were usually males (p<0.005), older than controls (p<0.0001), smokers (p=0.01), with a greater frequency of MUC5B rs35705950 (OR 3.5, 95% CI 1.3-9.4; p=0.01), and reduced diffusing capacity of the lung for carbon monoxide and oxygen saturation. Resistin, IL-6, SP-D, MMP-1, MMP-7 and MMP-13 were significantly increased in individuals with ILAs. Resistin (12±5 versus 9±4â ng·mL-1; p=0.0005) and MMP-13 (357±143 versus 298±116â pg·mL-1; p=0.004) were the most increased biomarkers. On follow-up (24±18â months), 18 individuals showed progression which was associated with gastro-oesophageal reflux disease (OR 4.1, 95% CI 1.2-12.9; p=0.02) and in females with diabetes mellitus (OR 5.3, 95% CI 1.0-27.4; p=0.01). CONCLUSIONS: Around 10% of respiratory asymptomatic individuals enrolled in our lung ageing programme show ILAs. Increased serum concentrations of pro-inflammatory molecules and MMPs are associated with ILAs.
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Doenças Pulmonares Intersticiais , Feminino , Humanos , Pulmão/diagnóstico por imagem , Masculino , Metaloproteinase 7 da Matriz , Mucina-5B , Fatores de RiscoRESUMO
A hypoxic microenvironment is a hallmark in different types of tumors; this phenomenon participates in a metabolic alteration that confers resistance to treatments. Because of this, it was proposed that a combination of 2-methoxyestradiol (2-ME) and sodium dichloroacetate (DCA) could reduce this alteration, preventing proliferation through the reactivation of aerobic metabolism in lung adenocarcinoma cell line (A549). A549 cells were cultured in a hypoxic chamber at 1% O2 for 72 hours to determine the effect of this combination on growth, migration, and expression of hypoxia-inducible factors (HIFs) by immunofluorescence. The effect in the metabolism was evaluated by the determination of glucose/glutamine consumption and the lactate/glutamate production. The treatment of 2-ME (10 µM) in combination with DCA (40 mM) under hypoxic conditions showed an inhibitory effect on growth and migration. Notably, this reduction could be attributed to 2-ME, while DCA had a predominant effect on metabolic activity. Moreover, this combination decreases the signaling of HIF-3α and partially HIF-1α but not HIF-2α. The results of this study highlight the antitumor activity of the combination of 2-ME 10 µl/DCA 40 mM, even in hypoxic conditions.
Assuntos
2-Metoxiestradiol/uso terapêutico , Antineoplásicos/farmacologia , Protocolos de Quimioterapia Combinada Antineoplásica/uso terapêutico , Carcinoma Pulmonar de Células não Pequenas/tratamento farmacológico , Ácido Dicloroacético/uso terapêutico , Neoplasias Pulmonares/tratamento farmacológico , Hipóxia Tumoral , Microambiente Tumoral , 2-Metoxiestradiol/farmacologia , Células A549 , Protocolos de Quimioterapia Combinada Antineoplásica/farmacologia , Proteínas Reguladoras de Apoptose/metabolismo , Fatores de Transcrição Hélice-Alça-Hélice Básicos/metabolismo , Carcinoma Pulmonar de Células não Pequenas/patologia , Movimento Celular/efeitos dos fármacos , Proliferação de Células/efeitos dos fármacos , Ácido Dicloroacético/farmacologia , Glucose/metabolismo , Ácido Glutâmico/metabolismo , Glutamina/metabolismo , Glicólise/efeitos dos fármacos , Humanos , Subunidade alfa do Fator 1 Induzível por Hipóxia/metabolismo , Ácido Láctico/metabolismo , Neoplasias Pulmonares/patologia , Proteínas Repressoras/metabolismo , Transdução de Sinais/efeitos dos fármacos , Hipóxia Tumoral/efeitos dos fármacos , Microambiente Tumoral/efeitos dos fármacos , Cicatrização/efeitos dos fármacosRESUMO
Idiopathic pulmonary fibrosis is a chronic and progressive disease of unknown cause. It is characterized by the aberrant activation of the bronchioalveolar epithelium, the formation of fibroblast foci and the excessive production extracellular matrix. The cellular and molecular mechanisms that contribute to the pathobiology of the disease are unclear. The CX3CL1-CX3CR1 axis regulates cellular responses that are known to be relevant in IPF, such as proliferation and collagen production. In this study, we characterize for the first time the expression of CX3CL1 and its receptor in lung tissue from patients with IPF; and its effect on collagen production in IPF fibroblasts. We found that CX3CL1-CX3CR1 axis has a modified expression in the lung tissue, importantly this axis is expressed on fibroblasts, and CX3CL1 decreased the collagen production in pulmonary fibroblasts derived from IPF patients.
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Receptor 1 de Quimiocina CX3C/metabolismo , Quimiocina CX3CL1/metabolismo , Colágeno/metabolismo , Fibrose Pulmonar Idiopática/patologia , Pulmão/patologia , Receptor 1 de Quimiocina CX3C/análise , Linhagem Celular , Proliferação de Células , Quimiocina CX3CL1/análise , Colágeno/análise , Matriz Extracelular/patologia , Fibroblastos/metabolismo , Humanos , Imuno-Histoquímica , Pulmão/citologia , Cultura Primária de Células , Transdução de SinaisRESUMO
BACKGROUND: Several lung structural and functional abnormalities may occur associated with aging, including emphysema. In this study, we evaluated the frequency and risk factors associated with emphysema in respiratory asymptomatic individuals enrolled in our Lung Aging Program. From a cohort of 687 subjects, we found by high-resolution computed tomography (HRCT) 29 individuals (4%) with emphysematous changes that were compared with 87 controls (3:1) randomly selected from the same cohort. METHODS: This was a transversal, observational, case-control study where we examined demographics and functional characteristics, as well as telomere length and serum Klotho concentration, two conditions that have been associated with aging and some aging-associated diseases including emphysema. RESULTS: Individuals with subclinical pulmonary emphysema were older (72 ± 9 versus 67 ± 6 years), and primarily smoker males with low body mass index. Despite that they were asymptomatic, two of them exhibited a decrease of forced expiratory volume in 1 s (FEV1), with a lower FEV1/FVC suggesting airway obstruction. Cigarette smoking (OR = 5.43, CI95% 1.8-16.7), family history of lung disease (OR = 4.32, CI95% 1.0-19.0) and lower body mass index (OR 7.22, CI95% 1.2-3.5) were risk factors for the development of lung emphysematous changes. No association was found with telomere length and Klotho serum concentration. CONCLUSION: Our findings reveal that a small but important percentage of older people without respiratory symptoms, present pulmonary emphysema and indicate that smoking exposure and genetic background may contribute to etiological factors.
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Envelhecimento , Pulmão/fisiopatologia , Enfisema Pulmonar/diagnóstico , Enfisema Pulmonar/fisiopatologia , Idoso , Idoso de 80 Anos ou mais , Estudos de Casos e Controles , Fumar Cigarros/efeitos adversos , Feminino , Glucuronidase/sangue , Humanos , Proteínas Klotho , Modelos Logísticos , Masculino , Pessoa de Meia-Idade , Análise Multivariada , Enfisema Pulmonar/sangue , Testes de Função Respiratória , Fatores de Risco , Telômero/fisiologia , Tomografia Computadorizada por Raios XRESUMO
Aging is the main risk factor for the development of idiopathic pulmonary fibrosis (IPF), a progressive and usually lethal lung disorder. Although the pathogenic mechanisms are uncertain, endoplasmic reticulum (ER) stress and impaired proteostasis that have been linked with aging are strongly associated with the pathogenesis of IPF. Using the Atg4b-deficient mice as a model, that partially reproduces the autophagy deficient conditions reported in aging and IPF lungs, we show for the first time how autophagy impairment and ER stress induction, contribute simultaneously to development of lung fibrosis in vivo. Increased expression of ER stress markers, inflammation and apoptosis of alveolar epithelial cells were observed in Atg4b-deficient mice compared to WT mice, when treated with the ER stress inducer tunicamycin. After tunicamycin treatment, Atg4b null lungs showed accumulation of its substrate LC3-I, demonstrating that these mice failed to induce autophagy despite the ER stress conditions. We also showed that compromised autophagy in lungs from Atg4b null mice is associated with exacerbated lung damage, epithelial apoptosis and the development of lung fibrosis at 21 days after tunicamycin treatment. Our findings indicate that ATG4B protein and autophagy are essential to mitigate ER stress and to prevent tunicamycin-induced epithelial apoptosis and lung fibrosis.
Assuntos
Proteínas Relacionadas à Autofagia/metabolismo , Autofagia/efeitos dos fármacos , Cisteína Endopeptidases/metabolismo , Retículo Endoplasmático/fisiologia , Células Epiteliais/fisiologia , Animais , Autofagia/fisiologia , Proteínas Relacionadas à Autofagia/genética , Linhagem Celular , Cisteína Endopeptidases/genética , Células Epiteliais/efeitos dos fármacos , Regulação da Expressão Gênica/efeitos dos fármacos , Pulmão/citologia , Mucosa Respiratória/citologia , Tunicamicina/farmacologiaRESUMO
Autophagy is a critical cellular homeostatic process that controls the turnover of damaged organelles and proteins. Impaired autophagic activity is involved in a number of diseases, including idiopathic pulmonary fibrosis suggesting that altered autophagy may contribute to fibrogenesis. However, the specific role of autophagy in lung fibrosis is still undefined. In this study, we show for the first time, how autophagy disruption contributes to bleomycin-induced lung fibrosis in vivo using an Atg4b-deficient mouse as a model. Atg4b-deficient mice displayed a significantly higher inflammatory response at 7 d after bleomycin treatment associated with increased neutrophilic infiltration and significant alterations in proinflammatory cytokines. Likewise, we found that Atg4b disruption resulted in augmented apoptosis affecting predominantly alveolar and bronchiolar epithelial cells. At 28 d post-bleomycin instillation Atg4b-deficient mice exhibited more extensive and severe fibrosis with increased collagen accumulation and deregulated extracellular matrix-related gene expression. Together, our findings indicate that the ATG4B protease and autophagy play a crucial role protecting epithelial cells against bleomycin-induced stress and apoptosis, and in the regulation of the inflammatory and fibrotic responses.
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Autofagia/efeitos dos fármacos , Bleomicina/farmacologia , Cisteína Endopeptidases/metabolismo , Homeostase/efeitos dos fármacos , Fibrose Pulmonar Idiopática/metabolismo , Animais , Apoptose/genética , Autofagia/fisiologia , Proteínas Relacionadas à Autofagia , Cisteína Endopeptidases/genética , Citocinas/genética , Citocinas/metabolismo , Células Epiteliais/citologia , Células Epiteliais/efeitos dos fármacos , Expressão Gênica/efeitos dos fármacos , Fibrose Pulmonar Idiopática/induzido quimicamente , Camundongos KnockoutRESUMO
BACKGROUND: Idiopathic pulmonary fibrosis (IPF) is a progressive and lethal disorder characterized by fibroproliferation and excessive accumulation of extracellular matrix in the lung. METHODS AND FINDINGS: Using oligonucleotide arrays, we identified osteopontin as one of the genes that significantly distinguishes IPF from normal lungs. Osteopontin was localized to alveolar epithelial cells in IPF lungs and was also significantly elevated in bronchoalveolar lavage from IPF patients. To study the fibrosis-relevant effects of osteopontin we stimulated primary human lung fibroblasts and alveolar epithelial cells (A549) with recombinant osteopontin. Osteopontin induced a significant increase of migration and proliferation in both fibroblasts and epithelial cells. Epithelial growth was inhibited by the pentapeptide Gly-Arg-Gly-Asp-Ser (GRGDS) and antibody to CD44, while fibroproliferation was inhibited by GRGDS and antibody to alphavbeta3 integrin. Fibroblast and epithelial cell migration were inhibited by GRGDS, anti-CD44, and anti-alphavbeta3. In fibroblasts, osteopontin up-regulated tissue inhibitor of metalloprotease-1 and type I collagen, and down-regulated matrix metalloprotease-1 (MMP-1) expression, while in A549 cells it caused up-regulation of MMP-7. In human IPF lungs, osteopontin colocalized with MMP-7 in alveolar epithelial cells, and application of weakest link statistical models to microarray data suggested a significant interaction between osteopontin and MMP-7. CONCLUSIONS: Our results provide a potential mechanism by which osteopontin secreted from the alveolar epithelium may exert a profibrotic effect in IPF lungs and highlight osteopontin as a potential target for therapeutic intervention in this incurable disease.
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Pulmão/metabolismo , Fibrose Pulmonar/metabolismo , Sialoglicoproteínas/metabolismo , Líquido da Lavagem Broncoalveolar/química , Movimento Celular , Proliferação de Células , Células Cultivadas , Células Epiteliais/efeitos dos fármacos , Células Epiteliais/metabolismo , Matriz Extracelular/metabolismo , Feminino , Perfilação da Expressão Gênica , Humanos , Masculino , Metaloproteinase 1 da Matriz/genética , Metaloproteinase 1 da Matriz/metabolismo , Metaloproteinase 7 da Matriz/metabolismo , Pessoa de Meia-Idade , Dados de Sequência Molecular , Osteopontina , Fibrose Pulmonar/patologia , Proteínas Recombinantes/farmacologia , Sialoglicoproteínas/farmacologia , Inibidor Tecidual de Metaloproteinase-1/genética , Inibidor Tecidual de Metaloproteinase-1/metabolismo , Regulação para CimaRESUMO
Objetivo: Describir la curva de alerta en parturientas de la altura. Diseño: Se realiza un estudio descriptivo, explicativo y prospectivo de corte transversal de enero a mayo de 1997. Pacientes: 400 parturientas nulíparas y multíparas a término, con presentación cefálica atendidas en el Hospital Víctor Ramos Guardia de Huaraz, seleccionadas con criterios de inclusión y que cursaron con trabajo de parto normal sin uso medicamentoso pre e intra parto. Fueron clasificadas en cuatro grupos de acuerdo al CLAP, cuidando de trazar la curva de alerta a partir de 4 cm de dilatación . El análisis estadístico, procedimiento de datos y la obtención de resultados con programas de EPIINFO 5, calculando el percentil 10 y construyéndose la curva a la izquierda con respecto a la curva del CLAP y aún más a la izquierda si comparamos con la curva a nivel del mar (HAMA), atribuyéndose este comportamiento al estado nutricional y al umbral doloroso de mujeres de la altura. Conclusión: La curva de alerta en parturientas de la altura presenta tiempos menores de dilatación. Palabras clave: Partograma, gestantes en altura.