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KEY POINTS: Ageing is associated with increased systemic inflammation and metabolic dysfunction that contributes to the development of age-associated diseases. The role of adipose tissue in immunometabolic alterations that take place with ageing is unknown in humans. We show, in healthy, active and lean older adults, that adipose tissue, but not skeletal muscle, displays considerable pro-inflammatory transcriptomic, cellular and secretory changes, as well as a reduction in insulin signalling proteins compared to younger adults. These findings indicate that adipose tissue undergoes substantial immunometabolic alterations with ageing, and that these changes are tissue-specific and more profound than those observed in skeletal muscle or in the circulation. These results identify adipose tissue as an important tissue in the biological ageing process in humans, which may exhibit signs of immunometabolic dysfunction prior to systemic manifestation. ABSTRACT: Ageing and obesity are both characterized by inflammation and a deterioration in metabolic health. It is now clear that adipose tissue plays a major role in inflammation and metabolic control in obesity, although little is known about the role of adipose tissue in human ageing. To understand how ageing impacts adipose tissue, we characterized subcutaneous adipose tissue and skeletal muscle samples from twelve younger (27 ± 4 years [Young]) and twelve older (66 ± 5 years [Old]) active/non-obese males. We performed a wide-range of whole-body and tissue measures, including RNA-sequencing and multicolour flow cytometry. We also measured a range of inflammatory and metabolic proteins in the circulation and their release by adipose tissue, ex vivo. Both adipose tissue and muscle had â¼2-fold more immune cells per gram of tissue with ageing. In adipose tissue, this immune cell infiltration was driven by increased memory/effector T-cells, whereas, in muscle, the accumulation was driven by memory/effector T-cells and macrophages. Transcriptomic analysis revealed that, with ageing, adipose tissue, but not muscle, was enriched for inflammatory transcripts/pathways related to acquired and innate immunity. Ageing also increased the adipose tissue pro-inflammatory secretory profile. Insulin signalling protein content was reduced in adipose tissue, but not muscle. Our findings indicate that adipose tissue undergoes substantial immunometabolic changes with ageing in humans, and that these changes are tissue-specific and more profound than those observed in the circulation and skeletal muscle.
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Resistência à Insulina , Tecido Adiposo/metabolismo , Idoso , Envelhecimento , Humanos , Masculino , Músculo Esquelético/metabolismo , Obesidade/metabolismoRESUMO
The last decade has seen an enormous increase in long non-coding RNA (lncRNA) research within rheumatology. LncRNAs are arbitrarily classed as non-protein encoding RNA transcripts that exceed 200 nucleotides in length. These transcripts have tissue and cell specific patterns of expression and are implicated in a variety of biological processes. Unsurprisingly, numerous lncRNAs are dysregulated in rheumatoid conditions, correlating with disease activity and cited as potential biomarkers and targets for therapeutic intervention. In this chapter, following an introduction into each condition, we discuss the lncRNAs involved in rheumatoid arthritis, osteoarthritis and systemic lupus erythematosus. These inflammatory joint conditions share several inflammatory signalling pathways and therefore not surprisingly many commonly dysregulated lncRNAs are shared across these conditions. In the interest of translational research only those lncRNAs which are strongly conserved have been addressed. The lncRNAs discussed here have diverse roles in regulating inflammation, proliferation, migration, invasion and apoptosis. Understanding the molecular basis of lncRNA function in rheumatology will be crucial in fully determining the inflammatory mechanisms that drive these conditions.
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Artrite Reumatoide , Lúpus Eritematoso Sistêmico , RNA Longo não Codificante , Reumatologia , Apoptose/genética , Artrite Reumatoide/genética , Humanos , Lúpus Eritematoso Sistêmico/genética , RNA Longo não Codificante/genéticaRESUMO
Idiopathic pulmonary fibrosis (IPF) is a progressive chronic disease characterized by excessing scarring of the lungs leading to irreversible decline in lung function. The aetiology and pathogenesis of the disease are still unclear, although lung fibroblast and epithelial cell activation, as well as the secretion of fibrotic and inflammatory mediators, have been strongly associated with the development and progression of IPF. Significantly, long non-coding RNAs (lncRNAs) are emerging as modulators of multiple biological processes, although their function and mechanism of action in IPF is poorly understood. LncRNAs have been shown to be important regulators of several diseases and their aberrant expression has been linked to the pathophysiology of fibrosis including IPF. This review will provide an overview of this emerging role of lncRNAs in the development of IPF.
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Suscetibilidade a Doenças , Fibrose Pulmonar Idiopática/etiologia , Fibrose Pulmonar Idiopática/metabolismo , RNA Longo não Codificante/genética , Células Epiteliais Alveolares/metabolismo , Células Epiteliais Alveolares/patologia , Animais , Biomarcadores , Cromatina/genética , Cromatina/metabolismo , Montagem e Desmontagem da Cromatina , Matriz Extracelular/metabolismo , Regulação da Expressão Gênica , Predisposição Genética para Doença , Humanos , Fibrose Pulmonar Idiopática/diagnóstico , Fibrose Pulmonar Idiopática/tratamento farmacológico , Pneumonia/complicações , Pneumonia/etiologia , Pneumonia/metabolismo , Pneumonia/patologia , Processamento Pós-Transcricional do RNA , Mucosa Respiratória/metabolismo , Mucosa Respiratória/patologia , Avaliação de Sintomas , Transcrição GênicaRESUMO
BACKGROUND: The mechanism underlying nonsevere and severe asthma remains unclear, although it is commonly associated with increased airway smooth muscle (ASM) mass. Long noncoding RNAs (lncRNAs) are known to be important in regulating healthy primary airway smooth muscle cells (ASMCs), whereas changed expression has been observed in CD8 T cells from patients with severe asthma. METHODS: Primary ASMCs were isolated from healthy subjects (n = 9) and patients classified as having nonsevere (n = 9) or severe (n = 9) asthma. ASMCs were exposed to dexamethasone and FCS. mRNA and lncRNA expression was measured by using a microarray and quantitative real-time PCR. Bioinformatic analysis was used to examine relevant biological pathways. Finally, the lncRNA plasmacytoma variant translocation 1 (PVT1) was inhibited by transfection of primary ASMCs with small interfering RNAs, and the effect on ASMC phenotype was examined. RESULTS: The mRNA expression profile was significantly different between patient groups after exposure to dexamethasone and FCS, and these were associated with biological pathways that might be relevant to the pathogenesis of asthma, including cellular proliferation and pathways associated with glucocorticoid activity. We also observed a significant change in lncRNA expression, yet the expression of only one lncRNA (PVT1) is decreased in patients with corticosteroid-sensitive nonsevere asthma and increased in patients with corticosteroid-insensitive severe asthma. Subsequent targeting studies demonstrated the importance of this lncRNA in controlling both proliferation and IL-6 release in ASMCs from patients with severe asthma. CONCLUSIONS: lncRNAs are associated with the aberrant phenotype observed in ASMCs from asthmatic patients. Targeting PVT1 might be effective in reducing airway remodeling in asthmatic patients.
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Asma/genética , Miócitos de Músculo Liso/metabolismo , RNA Longo não Codificante/metabolismo , Adulto , Asma/metabolismo , Asma/fisiopatologia , Feminino , Humanos , Interleucina-6/genética , Masculino , Pessoa de Meia-Idade , Análise de Sequência com Séries de Oligonucleotídeos , Fenótipo , Proteínas Proto-Oncogênicas c-myc/genética , RNA Longo não Codificante/genética , RNA Mensageiro/metabolismo , RNA Interferente Pequeno/genética , Transcrição Gênica , Transcriptoma , Adulto JovemRESUMO
BACKGROUND: Preinvasive squamous cell cancer (PSCC) are local transformations of bronchial epithelia that are frequently observed in current or former smokers. Their different grades and sizes suggest a continuum of dysplastic change with increasing severity, which may culminate in invasive squamous cell carcinoma (ISCC). As a consequence of the difficulty in isolating cancerous cells from biopsies, the molecular pathology that underlies their histological variability remains largely unknown. METHOD: To address this issue, we have employed microdissection to isolate normal bronchial epithelia and cancerous cells from low- and high-grade PSCC and ISCC, from paraffin embedded (FFPE) biopsies and determined gene expression using Affymetric Human Exon 1.0 ST arrays. Tests for differential gene expression were performed using the Bioconductor package limma followed by functional analyses of differentially expressed genes in IPA. RESULTS: Examination of differential gene expression showed small differences between low- and high-grade PSCC but substantial changes between PSCC and ISCC samples (184 vs 1200 p-value <0.05, fc ±1.75). However, the majority of the differentially expressed PSCC genes (142 genes: 77%) were shared with those in ISCC samples. Pathway analysis showed that these shared genes are associated with DNA damage response, DNA/RNA metabolism and inflammation as major biological themes. Cluster analysis identified 12 distinct patterns of gene expression including progressive up or down-regulation across PSCC and ISCC. Pathway analysis of incrementally up-regulated genes revealed again significant enrichment of terms related to DNA damage response, DNA/RNA metabolism, inflammation, survival and proliferation. Altered expression of selected genes was confirmed using RT-PCR, as well as immunohistochemistry in an independent set of 45 ISCCs. CONCLUSIONS: Gene expression profiles in PSCC and ISCC differ greatly in terms of numbers of genes with altered transcriptional activity. However, altered gene expression in PSCC affects canonical pathways and cellular and biological processes, such as inflammation and DNA damage response, which are highly consistent with hallmarks of cancer.
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Carcinoma de Células Escamosas/metabolismo , Carcinoma de Células Escamosas/patologia , Neoplasias Pulmonares/metabolismo , Neoplasias Pulmonares/patologia , Proteínas de Neoplasias/metabolismo , Fatores de Transcrição/metabolismo , Transcriptoma , Idoso , Células Cultivadas , Feminino , Perfilação da Expressão Gênica , Regulação Neoplásica da Expressão Gênica , Humanos , Microdissecção e Captura a Laser , Masculino , Pessoa de Meia-Idade , Invasividade Neoplásica , Células Tumorais CultivadasRESUMO
It is increasingly clear that long non-coding RNAs (lncRNAs) regulate a variety biological responses, and that they do so by a diverse range of mechanisms. In the field of immunology, recent publications have shown widespread changes in the expression of lncRNAs during the activation of the innate immune response and T cell development, differentiation, and activation. These lncRNAs control important aspects of immunity such as production of inflammatory mediators, differentiation, and cell migration through regulating protein-protein interactions or via their ability to basepair with RNA and DNA. We review the current understanding of the mechanism of action of these immune-related lncRNAs, discuss their impact on physiological and pathological processes, and highlight important areas of inquiry at the intersection between immunology and lncRNA biology.
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RNA Longo não Codificante/imunologia , Linfócitos T/imunologia , Imunidade Adaptativa , Animais , Diferenciação Celular , Movimento Celular , Humanos , Imunidade Inata , Mediadores da Inflamação/metabolismo , Ativação LinfocitáriaRESUMO
In chronic obstructive pulmonary disease (COPD), oxidative stress regulates the inflammatory response of bronchial epithelium and monocytes/macrophages through kinase modulation and has been linked to glucocorticoid unresponsiveness. Glycogen synthase-3ß (GSK3ß) inactivation plays a key role in mediating signaling processes upon reactive oxygen species (ROS) exposure. We hypothesized that GSK3ß is involved in oxidative stress-induced glucocorticoid insensitivity in COPD. We studied levels of phospho-GSK3ß-Ser9, a marker of GSK3ß inactivation, in lung sections and cultured monocytes and bronchial epithelial cells of COPD patients, control smokers, and nonsmokers. We observed increased levels of phospho-GSK3ß-Ser9 in monocytes, alveolar macrophages, and bronchial epithelial cells from COPD patients and control smokers compared with nonsmokers. Pharmacological inactivation of GSK3ß did not affect CXCL8 or granulocyte-macrophage colony-stimulating factor (GM-CSF) expression but resulted in glucocorticoid insensitivity in vitro in both inflammatory and structural cells. Further mechanistic studies in monocyte and bronchial epithelial cell lines showed that GSK3ß inactivation is a common effector of oxidative stress-induced activation of the MEK/ERK-1/2 and phosphatidylinositol 3-kinase/Akt signaling pathways leading to glucocorticoid unresponsiveness. In primary monocytes, the mechanism involved modulation of histone deacetylase 2 (HDAC2) activity in response to GSK3ß inactivation. In conclusion, we demonstrate for the first time that ROS-induced glucocorticoid unresponsiveness in COPD is mediated through GSK3ß, acting as a ROS-sensitive hub.
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Dexametasona/farmacologia , Glucocorticoides/farmacologia , Quinase 3 da Glicogênio Sintase/fisiologia , Doença Pulmonar Obstrutiva Crônica/enzimologia , Idoso , Células Cultivadas , Dexametasona/uso terapêutico , Feminino , Expressão Gênica/efeitos dos fármacos , Glucocorticoides/uso terapêutico , Glicogênio Sintase Quinase 3 beta , Histona Desacetilase 2/metabolismo , Humanos , Leucócitos Mononucleares/metabolismo , Macrófagos Alveolares/enzimologia , Masculino , Pessoa de Meia-Idade , Estresse Oxidativo , Doença Pulmonar Obstrutiva Crônica/tratamento farmacológico , Espécies Reativas de Oxigênio/metabolismo , Mucosa Respiratória/enzimologia , Transdução de SinaisRESUMO
BACKGROUND: The airway smooth muscle (ASM) cell maintains its own proliferative rate and contributes to the inflammatory response in the airways, effects that are inhibited by corticosteroids, used in the treatment of airways diseases. OBJECTIVE: We determined the differential expression of mRNAs, microRNAs (miRNAs) and long noncoding RNA species (lncRNAs) in primary ASM cells following treatment with a corticosteroid, dexamethasone, and fetal calf serum (FCS). METHODS: mRNA, miRNA and lncRNA expression was measured by microarray and quantitative real-time PCR. RESULTS: A small number of miRNAs (including miR-150, -371-5p, -718, -940, -1181, -1207-5p, -1915, and -3663-3p) were decreased following exposure to dexamethasone and FCS. The mRNA targets of these miRNAs were increased in expression. The changes in mRNA expression were associated with regulation of ASM actin cytoskeleton. We also observed changes in expression of lncRNAs, including natural antisense, pseudogenes, intronic lncRNAs, and intergenic lncRNAs following dexamethasone and FCS. We confirmed the change in expression of three of these, LINC00882, LINC00883, PVT1, and its transcriptional activator, c-MYC. We propose that four of these lincRNAs (RP11-46A10.4, LINC00883, BCYRN1, and LINC00882) act as miRNA 'sponges' for 4 miRNAs (miR-150, -371-5p, -940, -1207-5p). CONCLUSION: This in-vitro model of primary ASM cell phenotype was associated with the regulation of several ncRNAs. Their identification allows for in-vitro functional experimentation to establish causality with the primary ASM phenotype, and in airway diseases such as asthma and chronic obstructive pulmonary disease (COPD).
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Brônquios/citologia , Brônquios/fisiologia , Miócitos de Músculo Liso/citologia , Miócitos de Músculo Liso/fisiologia , RNA não Traduzido/fisiologia , Adulto , Células Cultivadas , Feminino , Humanos , Masculino , Pessoa de Meia-Idade , Cultura Primária de Células , Adulto JovemRESUMO
BACKGROUND: Although previous studies have implicated tissue CD4(+) T cells in the development and maintenance of the inflammatory response in asthmatic patients, little is known about the role of CD8(+) T cells. There is now accumulating evidence that microRNAs and other noncoding RNAs are important regulators of T-cell function. OBJECTIVES: We sought to use transcriptomics to determine the activation state of circulating CD4(+) and CD8(+) T cells in patients with nonsevere and severe asthma. METHODS: mRNA and noncoding RNA expression in circulating T cells was measured by means of microarray, quantitative real-time PCR, or both. RESULTS: Comparison of mRNA expression showed widespread changes in the circulating CD8(+) but not CD4(+) T cells from patients with severe asthma. No changes were observed in the CD4(+) and CD8(+) T cells in patients with nonsevere asthma versus those in healthy control subjects. Bioinformatics analysis showed that the changes in CD8(+) T-cell mRNA expression were associated with multiple pathways involved in T-cell activation. As with mRNAs, we also observed widespread changes in expression of noncoding RNA species, including natural antisense, pseudogenes, intronic long noncoding RNAs (lncRNAs), and intergenic lncRNAs in CD8(+) T cells from patients with severe asthma. Measurement of the microRNA expression profile showed selective downregulation of miR-28-5p in CD8(+) T cells and reduction of miR-146a and miR-146b in both CD4(+) and CD8(+) T cells. CONCLUSIONS: Severe asthma is associated with the activation of circulating CD8(+) T cells but not CD4(+) T cells. This response is correlated with the downregulation of miR-146a/b and miR-28-5p, as well as changes in the expression of multiple species of lncRNA that might regulate CD8(+) T-cell function.
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Asma/genética , Asma/imunologia , Linfócitos T CD8-Positivos/imunologia , Perfilação da Expressão Gênica , Ativação Linfocitária/genética , Adulto , Linfócitos T CD4-Positivos/imunologia , Feminino , Regulação da Expressão Gênica , Humanos , Masculino , MicroRNAs/metabolismo , Pessoa de Meia-Idade , RNA Mensageiro/metabolismo , RNA não Traduzido/metabolismo , Transdução de Sinais , Adulto JovemRESUMO
BACKGROUND: Osteoarthritis (OA), a multifaceted condition, poses a significant challenge for the successful clinical development of therapeutics due to heterogeneity. However, classifying molecular endotypes of OA pathogenesis could provide invaluable phenotype-directed routes for stratifying subgroups of patients for targeted therapeutics, leading to greater chances of success in trials. This study establishes endotypes in OA soft joint tissue driven by obesity in both load-bearing and non-load bearing joints. METHODS: Hand, hip, knee and foot joint synovial tissue was obtained from OA patients (n = 32) classified as obese (BMI > 30) or normal weight (BMI 18.5-24.9). Isolated fibroblasts (OA SF) were assayed by Olink proteomic panel, seahorse metabolic flux assay, Illumina's NextSeq 500 bulk and Chromium 10X single cell RNA-sequencing, validated by Luminex and immunofluorescence. RESULTS: Targeted proteomic, metabolic and transcriptomic analysis found the inflammatory landscape of OA SFs are independently impacted by obesity, joint loading and anatomical site with significant heterogeneity between obese and normal weight patients, confirmed by bulk RNAseq. Further investigation by single cell RNAseq identified four functional molecular endotypes including obesity specific subsets defined by an inflammatory endotype related to immune cell regulation, fibroblast activation and inflammatory signaling, with up-regulated CXCL12, CFD and CHI3L1 expression. Luminex confirmed elevated chitase3-like-1(229.5 vs. 49.5 ng/ml, p < .05) and inhibin (20.6 vs. 63.8 pg/ml, p < .05) in obese and normal weight OA SFs, respectively. Lastly, we find SF subsets in obese patients spatially localise in sublining and lining layers of OA synovium and can be distinguished by differential expression of the transcriptional regulators MYC and FOS. CONCLUSION: These findings demonstrate the significance of obesity in changing the inflammatory landscape of synovial fibroblasts in both load bearing and non-load bearing joints. Describing multiple heterogeneous OA SF populations characterised by specific molecular endotypes, which drive heterogeneity in OA disease pathogenesis. These molecular endotypes may provide a route for the stratification of patients in clinical trials, providing a rational for the therapeutic targeting of specific SF subsets in specific patient populations with arthritic conditions.
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Osteoartrite , Proteômica , Humanos , Membrana Sinovial/metabolismo , Membrana Sinovial/patologia , Osteoartrite/metabolismo , Osteoartrite/patologia , Obesidade/genética , Obesidade/metabolismo , Fibroblastos/metabolismoRESUMO
CONTEXT: Adipose tissue and physical inactivity both influence metabolic health and systemic inflammation, but how adipose tissue responds to chronic physical inactivity is unknown. OBJECTIVE: This work aimed to characterize the impact of chronic physical inactivity on adipose tissue in healthy, young males. METHODS: We collected subcutaneous adipose tissue from 20 healthy, young men before and after 60 days of complete bed rest with energy intake reduced to maintain energy balance and fat mass. We used RNA sequencing, flow cytometry, ex vivo tissue culture, and targeted protein analyses to examine adipose tissue phenotype. RESULTS: Our results indicate that the adipose tissue transcriptome, stromal cellular compartment, and insulin signaling protein abundance are largely unaffected by bed rest when fat mass is kept stable. However, there was an increase in the circulating concentration of several adipokines, including plasma leptin, which was associated with inactivity-induced increases in plasma insulin and absent from adipose tissue cultured ex vivo under standardized culture conditions. CONCLUSION: Physical inactivity-induced disturbances to adipokine concentrations such as leptin, without changes to fat mass, could have profound metabolic implications outside a clinical facility when energy intake is not tightly controlled.
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Metabolismo Basal/imunologia , Comportamento Sedentário , Gordura Subcutânea/metabolismo , Adulto , Repouso em Cama , Voluntários Saudáveis , Humanos , Inflamação/sangue , Inflamação/imunologia , Inflamação/metabolismo , Leptina/sangue , Leptina/metabolismo , Masculino , Pessoa de Meia-Idade , Gordura Subcutânea/imunologia , Adulto JovemRESUMO
MicroRNAs are extremely promising candidates for early cancer diagnosis and prognosis. The levels of circulating microRNAs provide valuable information about cancer disease at its early stages. However, the levels of microRNAs that need to be detected are extremely low and difficult to discriminate from a large pool of oligonucleotides. There is the need for accurate, rapid and sensitive detection methodologies for detection of microRNAs. We developed electrochemical impedance spectroscopy peptide nucleic acid (PNA)-based sensors that can detect miRNAs in diluted serum with a limit of detection of 0.38 fM. In order to further improve the accuracy and reliability of the sensors, we developed an assay using magnetic beads for simple and rapid fishing of target microRNAs from solution and its pre-concentration prior to electrochemical detection. Our methodology utilizes magnetic beads for the capture of the target microRNA from solution and brings the concentrated sample to the sensor surface. We modify the magnetic beads with locked nucleic acids (LNA), which have high affinity and specificity to their complementary microRNA sequence. The separated and concentrated microRNA is then detected using the PNA-based sensors. By exposing the sensing electrodes only to the captured microRNAs, interferences from other nucleotides or biomolecules from the sample are eliminated.
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Técnicas Biossensoriais , MicroRNA Circulante/genética , Técnicas Eletroquímicas , MicroRNAs/genética , Hibridização de Ácido Nucleico/métodos , Oligonucleotídeos , Espectroscopia Dielétrica , Humanos , Espectrofotometria UltravioletaRESUMO
Osteoarthritis (OA) and rheumatoid arthritis (RA) are two of the most common chronic inflammatory joint diseases, for which there remains a great clinical need to develop safer and more efficacious pharmacological treatments. The pathology of both OA and RA involves multiple tissues within the joint, including the synovial joint lining and the bone, as well as the articular cartilage in OA. In this review, we discuss the potential for the development of oligonucleotide therapies for these disorders by examining the evidence that oligonucleotides can modulate the key cellular pathways that drive the pathology of the inflammatory diseased joint pathology, as well as evidence in preclinical in vivo models that oligonucleotides can modify disease progression.
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BACKGROUND: Synovial inflammation is associated with pain severity in patients with knee osteoarthritis (OA). The aim here was to determine in a population with knee OA, whether synovial tissue from areas associated with pain exhibited different synovial fibroblast subsets, compared to synovial tissue from sites not associated with pain. A further aim was to compare differences between early and end-stage disease synovial fibroblast subsets. METHODS: Patients with early knee OA (n = 29) and end-stage knee OA (n = 22) were recruited. Patient reported pain was recorded by questionnaire and using an anatomical knee pain map. Proton density fat suppressed MRI axial and sagittal sequences were analysed and scored for synovitis. Synovial tissue was obtained from the medial and lateral parapatellar and suprapatellar sites. Fibroblast single cell RNA sequencing was performed using Chromium 10X and analysed using Seurat. Transcriptomes were functionally characterised using Ingenuity Pathway Analysis and the effect of fibroblast secretome on neuronal growth assessed using rat DRGN. FINDINGS: Parapatellar synovitis was significantly associated with the pattern of patient-reported pain in knee OA patients. Synovial tissue from sites of patient-reported pain exhibited a differential transcriptomic phenotype, with distinct synovial fibroblast subsets in early OA and end-stage OA. Functional pathway analysis revealed that synovial tissue and fibroblast subsets from painful sites promoted fibrosis, inflammation and the growth and activity of neurons. The secretome of fibroblasts from early OA painful sites induced greater survival and neurite outgrowth in dissociated adult rodent dorsal root ganglion neurons. INTERPRETATION: Sites of patient-reported pain in knee OA exhibit a different synovial tissue phenotype and distinct synovial fibroblast subsets. Further interrogation of these fibroblast pathotypes will increase our understanding of the role of synovitis in OA joint pain and provide a rationale for the therapeutic targeting of fibroblast subsets to alleviate pain in patients. FUNDING: This study was funded by Versus Arthritis, UK (21530; 21812).
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Artralgia/patologia , Fibroblastos/patologia , Articulação do Joelho/patologia , Osteoartrite do Joelho/patologia , Idoso , Feminino , Humanos , Inflamação/patologia , Masculino , Pessoa de Meia-Idade , Dor/patologia , Medição da Dor/métodos , Fenótipo , Secretoma/fisiologia , Índice de Gravidade de Doença , Membrana Sinovial/patologia , Sinovite/patologiaRESUMO
BACKGROUND: Idiopathic pulmonary fibrosis (IPF) is a fatal lung disease accounting for 1% of UK deaths. In the familial form of pulmonary fibrosis, causal genes have been identified in about 30% of cases, and a majority of these causal genes are associated with telomere maintenance. Prematurely shortened leukocyte telomere length is associated with IPF and chronic obstructive pulmonary disease (COPD), a disease with similar demographics and shared risk factors. Using mendelian randomisation, we investigated evidence supporting a causal role for short telomeres in IPF and COPD. METHODS: Mendelian randomisation inference of telomere length causality was done for IPF (up to 1369 cases) and COPD (13â538 cases) against 435â866 controls of European ancestry in UK Biobank. Polygenic risk scores were calculated and two-sample mendelian randomisation analyses were done using seven genetic variants previously associated with telomere length, with replication analysis in an IPF cohort (2668 cases vs 8591 controls) and COPD cohort (15â256 cases vs 47â936 controls). FINDINGS: In the UK Biobank, a genetically instrumented one-SD shorter telomere length was associated with higher odds of IPF (odds ratio [OR] 4·19, 95% CI 2·33-7·55; p=0·0031) but not COPD (1·07, 0·88-1·30; p=0·51). Similarly, an association was found in the IPF replication cohort (12·3, 5·05-30·1; p=0·0015) and not in the COPD replication cohort (1·04, 0·71-1·53; p=0·83). Meta-analysis of the two-sample mendelian randomisation results provided evidence inferring that shorter telomeres cause IPF (5·81 higher odds of IPF, 95% CI 3·56-9·50; p=2·19â×â10-12). There was no evidence to infer that telomere length caused COPD (OR 1·07, 95% CI 0·90-1·27; p=0·46). INTERPRETATION: Cellular senescence is hypothesised as a major driving force in IPF and COPD; telomere shortening might be a contributory factor in IPF, suggesting divergent mechanisms in COPD. Defining a key role for telomere shortening enables greater focus in telomere-related diagnostics, treatments, and the search for a cure in IPF. Investigation of therapies that improve telomere length is warranted. FUNDING: Medical Research Council.
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Fibrose Pulmonar Idiopática/genética , Doença Pulmonar Obstrutiva Crônica/genética , Encurtamento do Telômero/genética , Idoso , Estudos de Casos e Controles , Causalidade , Feminino , Humanos , Fibrose Pulmonar Idiopática/epidemiologia , Masculino , Análise da Randomização Mendeliana , Pessoa de Meia-Idade , Doença Pulmonar Obstrutiva Crônica/epidemiologia , Fatores de RiscoRESUMO
BACKGROUND: Despite the widespread induction of miR-146a during the innate immune response little is known regarding its biogenesis, function and mechanism. We have therefore examined the role of miR-146a during the interleukin (IL)-1beta-stimulated IL-6 and IL-8 release and proliferation in primary human airway smooth muscle (HASM) cells. METHODS: HASM cells were isolated from human lung re-section, cultured to a maximum of 3 - 6 passages and then exposed to IL-1beta. miR-146a expression were determined by qRT-PCR, IL-6 and IL-8 release by ELISA and proliferation using bromodeoxyuridine incorporation. The role of NF-kappaB and the MAP kinase pathways was assessed using pharmacological inhibitors of IKK2 (TPCA-1), JNK (SP600125), p38 MAP kinase (SB203580) and MEK-1/2 (PD98059). miR-146a function was determined following transfection of HASM with inhibitors and mimics using Amaxa electroporation. RESULTS: IL-1beta induced a time-dependent and prolonged 100-fold induction in miR-146a expression, which correlated with release of IL-6 and IL-8. Exposure to IL-1beta had no effect upon HASM proliferation. Pharmacological studies showed that expression of primary miR-146a was regulated at the transcriptional levels by NF-kappaB whilst post-transcriptional processing to mature miR-146a was regulated by MEK-1/2 and JNK-1/2. Functional studies indicated that IL-1beta-induced miR-146a expression does not negatively regulate IL-6 and IL-8 release or basal proliferation. However, inhibition of IL-1beta-induced IL-6 and IL-8 release was observed at the super-maximal intracellular miR-146a levels obtained by transfection with miR-146a mimics and indicates that studies using miRNA mimics can produce false positive results. Mechanistic studies showed that in the presence of super-maximal levels, the action of miR-146a mimics was mediated at a step following IL-6 and IL-8 mRNA transcription and not through down-regulation of IL-1 receptor associated kinase 1 (IRAK-1) and TNF receptor-associated factor 6 (TRAF6) protein expression, two predicted miR-146a targets involved in IL-1beta signalling. CONCLUSIONS: We have shown that IL-1beta-induced miR-146a expression in HASM and that this was regulated at the transcriptional level by NF-kappaB and at the post-transcriptional level by the MEK-1/2 and JNK-1/2. Unlike previous reports, studies using miRNA inhibitors showed that miR-146a expression did not regulate IL-6 and IL-8 release or proliferation and suggest miR-146a function and mechanism is cell-type dependent.
Assuntos
Imunidade Inata , Interleucina-1beta/metabolismo , Pulmão/metabolismo , MicroRNAs/metabolismo , Miócitos de Músculo Liso/metabolismo , Proliferação de Células , Células Cultivadas , Relação Dose-Resposta a Droga , Ensaio de Imunoadsorção Enzimática , Humanos , Quinase I-kappa B/antagonistas & inibidores , Quinase I-kappa B/metabolismo , Interleucina-6/metabolismo , Interleucina-8/metabolismo , Proteínas Quinases JNK Ativadas por Mitógeno/antagonistas & inibidores , Proteínas Quinases JNK Ativadas por Mitógeno/metabolismo , Pulmão/efeitos dos fármacos , Pulmão/imunologia , MAP Quinase Quinase 1/antagonistas & inibidores , MAP Quinase Quinase 1/metabolismo , MAP Quinase Quinase 2/antagonistas & inibidores , MAP Quinase Quinase 2/metabolismo , Miócitos de Músculo Liso/efeitos dos fármacos , Miócitos de Músculo Liso/imunologia , NF-kappa B/antagonistas & inibidores , NF-kappa B/metabolismo , Reação em Cadeia da Polimerase , Inibidores de Proteínas Quinases/farmacologia , Processamento Pós-Transcricional do RNA , Fatores de Tempo , Transcrição Gênica , Transfecção , Regulação para Cima , Proteínas Quinases p38 Ativadas por Mitógeno/antagonistas & inibidores , Proteínas Quinases p38 Ativadas por Mitógeno/metabolismoRESUMO
OBJECTIVE: To identify long noncoding RNAs (lncRNAs) associated with the inflammatory phenotype of synovial fibroblasts from obese patients with osteoarthritis (OA), and to explore the expression and function of these lncRNAs. METHODS: Synovium was collected from normal-weight patients with hip fracture (non-OA; n = 6) and from normal-weight (n = 8) and obese (n = 8) patients with hip OA. Expression of RNA was determined by RNA-sequencing and quantitative reverse transcription-polymerase chain reaction. Knockdown of lncRNA was performed using LNA-based GapmeRs. Synovial fibroblast cytokine production was measured by enzyme-linked immunosorbent assay. RESULTS: Synovial fibroblasts from obese patients with OA secreted greater levels of interleukin-6 (IL-6) (mean ± SEM 162 ± 21 pg/ml; P < 0.001) and CXCL8 (262 ± 67 pg/ml; P < 0.05) compared to fibroblasts from normal-weight patients with OA (IL-6, 51 ± 4 pg/ml; CXCL8, 78 ± 11 pg/ml) or non-OA patients (IL-6, 35 ± 3 pg/ml; CXCL8, 56 ± 6 pg/ml) (n = 6 patients per group). RNA-sequencing revealed that fibroblasts from obese OA patients exhibited an inflammatory transcriptome, with increased expression of proinflammatory messenger RNAs (mRNAs) as compared to that in fibroblasts from normal-weight OA or non-OA patients (>2-fold change, P < 0.05; n = 4 patients per group). A total of 19 lncRNAs were differentially expressed between normal-weight OA and non-OA patient fibroblasts, and a further 19 lncRNAs were differentially expressed in fibroblasts from obese OA patients compared to normal-weight OA patients (>2-fold change, P < 0.05 for each), which included the lncRNA for metastasis-associated lung adenocarcinoma transcript 1 (MALAT1). MALAT1 was rapidly induced upon stimulation of OA synovial fibroblasts with proinflammatory cytokines, and was up-regulated in the synovium from obese OA patients as compared to normal-weight OA patients (1.6-fold change, P < 0.001) or non-OA patients (6-fold change, P < 0.001). MALAT1 knockdown in OA synovial fibroblasts (n = 4 patients) decreased the levels of mRNA expression and protein secretion of CXCL8 (>1.5-fold change, P < 0.01), whereas it increased expression of mRNAs for TRIM6 (>2-fold change, P < 0.01), IL7R (<2-fold change, P < 0.01), HIST1H1C (>1.5-fold change, P < 0.001), and MAML3 (>1.5-fold change, P < 0.001). In addition, MALAT1 knockdown inhibited the proliferation of synovial fibroblasts from obese patients with OA. CONCLUSION: Synovial fibroblasts from obese patients with hip OA exhibit an inflammatory phenotype. MALAT1 lncRNA may mediate joint inflammation in obese OA patients.
Assuntos
Fibroblastos/metabolismo , Interleucina-6/metabolismo , Obesidade/metabolismo , Osteoartrite do Quadril/metabolismo , RNA Longo não Codificante/metabolismo , Membrana Sinovial/metabolismo , Idoso , Proliferação de Células/fisiologia , Feminino , Humanos , Inflamação/metabolismo , Masculino , Pessoa de Meia-Idade , Obesidade/complicações , Osteoartrite do Quadril/complicaçõesRESUMO
Innate immunity provides the initial defence against infection and it is now clear that long non-coding RNAs (lncRNAs) are important regulators of this response. Following activation of the innate response, we commonly see rapid induction of these lncRNAs and this is often mediated via the pro-inflammatory transcription factor, nuclear factor-κB (NF-κB). Knockdown studies have shown that lncRNAs tend to act in trans to regulate the expression of multiple inflammatory mediators and other responses. Mechanistically, many lncRNAs have demonstrated acting through heterogeneous nuclear ribonucleoproteins, complexes that are implicated chromatin re-modelling, transcription process and translation. In addition, these lncRNAs have also been shown to interact with multiple other proteins involved in the regulation of chromatin re-modelling, as well as those proteins involved in intracellular immune signalling, which include NF-κB. In this review, we will describe the evidence that supports this emerging role of lncRNA in the innate immune response.
RESUMO
The N-methyl-D-aspartate (NMDA) receptor antagonist ketamine offers promising perspectives for the treatment of major depressive disorder. Although ketamine demonstrates rapid and long-lasting effects, even in treatment-resistant patients, to date, the underlying mode of action remains elusive. Thus, the aim of our study was to investigate the molecular mechanism of ketamine at clinically relevant concentrations by establishing an in vitro model based on human induced pluripotent stem cells (iPSCs)-derived neural progenitor cells (NPCs). Notably, ketamine increased the proliferation of NPCs independent of the NMDA receptor, while transcriptome analysis revealed significant upregulation of insulin-like growth factor 2 (IGF2) and p11, a member of the S100 EF-hand protein family, which are both implicated in the pathophysiology of depression, 24 h after ketamine treatment. Ketamine (1 µM) was able to increase cyclic adenosine monophosphate (cAMP) signaling in NPCs within 15 min and cell proliferation, while ketamine-induced IGF2 expression was reduced after PKA inhibition with cAMPS-Rp. Furthermore, 24 h post-administration of ketamine (15 mg/kg) in vivo confirmed phosphorylation of extracellular signal-regulated protein kinases 1 and 2 (ERK1/2) in the subgranular zone (SGZ) of the hippocampus in C57BL/6 mice. In conclusion, ketamine promotes the proliferation of NPCs presumably by involving cAMP-IGF2 signaling.