Assuntos
COVID-19/virologia , Interações entre Hospedeiro e Microrganismos/fisiologia , SARS-CoV-2/patogenicidade , Células A549 , COVID-19/imunologia , Linhagem Celular Tumoral , Células Epiteliais/imunologia , Células Epiteliais/virologia , Interações entre Hospedeiro e Microrganismos/imunologia , Humanos , Imunidade Inata/imunologia , Pulmão/imunologia , Pulmão/virologia , Proteômica/métodos , SARS-CoV-2/imunologiaRESUMO
Hyper-secretion and/or hyper-concentration of mucus is a defining feature of multiple obstructive lung diseases, including chronic obstructive pulmonary disease (COPD). Mucus itself is composed of a mixture of water, ions, salt and proteins, of which the gel-forming mucins, MUC5AC and MUC5B, are the most abundant. Recent studies have linked the concentrations of these proteins in sputum to COPD phenotypes, including chronic bronchitis (CB) and acute exacerbations (AE). We sought to determine whether common genetic variants influence sputum mucin concentrations and whether these variants are also associated with COPD phenotypes, specifically CB and AE. We performed a GWAS to identify quantitative trait loci for sputum mucin protein concentration (pQTL) in the Sub-Populations and InteRmediate Outcome Measures in COPD Study (SPIROMICS, n = 708 for total mucin, n = 215 for MUC5AC, MUC5B). Subsequently, we tested for associations of mucin pQTL with CB and AE using regression modeling (n = 822-1300). Replication analysis was conducted using data from COPDGene (n = 5740) and by examining results from the UK Biobank. We identified one genome-wide significant pQTL for MUC5AC (rs75401036) and two for MUC5B (rs140324259, rs10001928). The strongest association for MUC5B, with rs140324259 on chromosome 11, explained 14% of variation in sputum MUC5B. Despite being associated with lower MUC5B, the C allele of rs140324259 conferred increased risk of CB (odds ratio (OR) = 1.42; 95% confidence interval (CI): 1.10-1.80) as well as AE ascertained over three years of follow up (OR = 1.41; 95% CI: 1.02-1.94). Associations between rs140324259 and CB or AE did not replicate in COPDGene. However, in the UK Biobank, rs140324259 was associated with phenotypes that define CB, namely chronic mucus production and cough, again with the C allele conferring increased risk. We conclude that sputum MUC5AC and MUC5B concentrations are associated with common genetic variants, and the top locus for MUC5B may influence COPD phenotypes, in particular CB.
Assuntos
Mucinas , Doença Pulmonar Obstrutiva Crônica , Humanos , Mucinas/genética , Mucinas/metabolismo , Escarro/metabolismo , Doença Pulmonar Obstrutiva Crônica/genética , Muco/metabolismo , FenótipoRESUMO
Rationale: Microplastics are a pressing global concern, and inhalation of microplastic fibers has been associated with interstitial and bronchial inflammation in flock workers. However, how microplastic fibers affect the lungs is unknown. Objectives: Our aim was to assess the effects of 12 × 31 µm nylon 6,6 (nylon) and 15 × 52 µm polyethylene terephthalate (polyester) textile microplastic fibers on lung epithelial growth and differentiation. Methods: We used human and murine alveolar and airway-type organoids as well as air-liquid interface cultures derived from primary lung epithelial progenitor cells and incubated these with either nylon or polyester fibers or nylon leachate. In addition, mice received one dose of nylon fibers or nylon leachate, and, 7 days later, organoid-forming capacity of isolated epithelial cells was investigated. Measurements and Main Results: We observed that nylon microfibers, more than polyester, inhibited developing airway organoids and not established ones. This effect was mediated by components leaching from nylon. Epithelial cells isolated from mice exposed to nylon fibers or leachate also formed fewer airway organoids, suggesting long-lasting effects of nylon components on epithelial cells. Part of these effects was recapitulated in human air-liquid interface cultures. Transcriptomic analysis revealed upregulation of Hoxa5 after exposure to nylon fibers. Inhibiting Hoxa5 during nylon exposure restored airway organoid formation, confirming Hoxa5's pivotal role in the effects of nylon. Conclusions: These results suggest that components leaching from nylon 6,6 may especially harm developing airways and/or airways undergoing repair, and we strongly encourage characterization in more detail of both the hazard of and the exposure to microplastic fibers.
Assuntos
Caprolactama/análogos & derivados , Microplásticos , Plásticos , Polímeros , Camundongos , Humanos , Animais , Nylons , Têxteis , PoliésteresRESUMO
BACKGROUND: Airway remodeling is a prominent feature of asthma, which involves increased airway smooth muscle mass and altered extracellular matrix composition. Bronchial thermoplasty (BT), a bronchoscopic treatment for severe asthma, targets airway remodeling. OBJECTIVE: We sought to investigate the effect of BT on extracellular matrix composition and its association with clinical outcomes. METHODS: This is a substudy of the TASMA trial. Thirty patients with severe asthma were BT-treated, of whom 13 patients were treated for 6 months with standard therapy (control group) before BT. Demographic data, clinical data including pulmonary function, and bronchial biopsies were collected. Biopsies at BT-treated and nontreated locations were analyzed by histological and immunohistochemical staining. Associations between histology and clinical outcomes were explored. RESULTS: Six months after treatment, it was found that the reticular basement membrane thickness was reduced from 7.28 µm to 5.74 µm (21% relative reduction) and the percentage area of tissue positive for collagen increased from 26.3% to 29.8% (13% relative increase). Collagen structure analysis revealed a reduction in the curvature frequency of fibers. The percentage area positive for fibulin-1 and fibronectin increased by 2.5% and 5.9%, respectively (relative increase of 124% and 15%). No changes were found for elastin. The changes in collagen and fibulin-1 negatively associated with changes in FEV1 reversibility. CONCLUSIONS: Besides reduction of airway smooth muscle mass, BT has an impact on reticular basement membrane thickness and the extracellular matrix arrangement characterized by an increase in tissue area occupied by collagen with a less dense fiber organization. Both collagen and fibulin-1 are negatively associated with the change in FEV1 reversibility.
Assuntos
Asma , Termoplastia Brônquica , Humanos , Brônquios/cirurgia , Brônquios/patologia , Remodelação das Vias Aéreas , Asma/tratamento farmacológico , Matriz Extracelular/patologia , ColágenoRESUMO
The role of alternative splicing in chronic obstructive pulmonary disease (COPD) is still largely unknown. We aimed to investigate the differences in alternatively splicing events between patients with mild-to-moderate and severe COPD compared with non-COPD control subjects and to identify splicing factors associated with aberrant alternative splicing in COPD. For this purpose, we performed genome-wide RNA-sequencing analysis of bronchial brushings from 23 patients with mild-to-moderate COPD, 121 with severe COPD, and 23 non-COPD control subjects. We found a significant difference in the frequency of alternative splicing events in patients with mild-to-moderate and severe COPD compared with non-COPD control subjects. There were from two to eight times (depending on event type) more differential alternative splicing events in the severe than in the mild-to-moderate stage. The severe COPD samples showed less intron retention and more exon skipping. It is interesting that the transcript levels of the top 10 differentially expressed splicing factors were significantly correlated with the percentage of many alternatively spliced transcripts in severe COPD. The aberrant alternative splicing in severe COPD was predicted to increase the overall protein-coding capacity of gene products. In conclusion, we observed large and significant differences in alternative splicing between bronchial samples of patients with COPD and control subjects, with more events observed in severe than in mild-to-moderate COPD. The changes in the expression of several splicing factors correlated with prevalence of alternative splicing in severe COPD. Alternative splicing can indirectly impact gene expression by changing the relative abundance of protein-coding isoforms potentially influencing pathophysiological changes. The results provide a better understanding of COPD-related alternative splicing changes.
Assuntos
Processamento Alternativo , Doença Pulmonar Obstrutiva Crônica , Transcriptoma , Humanos , Doença Pulmonar Obstrutiva Crônica/genética , Processamento Alternativo/genética , Masculino , Feminino , Transcriptoma/genética , Idoso , Pessoa de Meia-Idade , Índice de Gravidade de Doença , Estudos de Casos e Controles , Éxons/genéticaRESUMO
IL-33 and IL-1RL1 are well-replicated asthma genes that act in a single pathway toward type-2 immune responses. IL-33 is expressed by basal epithelial cells, and the release of IL-33 upon epithelial damage can activate innate lymphoid cells, T helper-2 cells, basophilic granulocytes, and mast cells through a receptor complex containing IL-1RL1. However, it is unknown how bronchial epithelial cells respond to IL-33, and whether this response is increased in the disease. We aimed to characterize the IL-33-driven transcriptomic changes in cultured primary bronchial epithelial cells from patients with asthma and healthy controls. Primary bronchial epithelial cells (PBECs) were obtained by bronchial brushing from six healthy control for air-liquid interface (ALI) cultures, whereas we selected eight healthy controls and seven patients with asthma for epithelial organoid cultures. We then stimulated the cultures for 24 h with recombinant IL-33 (rhIL33) at various concentrations with 1, 10, and 50 ng/mL for the ALI cultures and 20 ng/mL and 100 ng/mL for the organoid cultures, followed by RNA-sequencing and differential gene expression analysis. We did not detect any genome-wide significant differentially expressed genes after stimulation of PBECs with IL-33, irrespective of growth in three-dimensional (3-D) epithelial organoids or after differentiation in ALI cultures. These results were identical between PBECs obtained from patients with asthma or from healthy control subjects. We detected very low levels of IL-1RL1 gene expression in these airway epithelial cell cultures. We conclude that bronchial epithelial cells do not have a transcriptional response to IL-33, independent of their differentiation state. Hence, the airway epithelium acts as a source of IL-33 but does not seem to contribute to the response upon release of the alarmin after epithelial damage.NEW & NOTEWORTHY The IL-33/IL-1RL1 pathway stands as a formidable genetic predisposition for asthma, with ongoing clinical developments of various drugs designed to mitigate its influence in patients with asthma. The absence of a transcriptomic reaction to IL-33 within the bronchial epithelium holds significance in the pursuit of identifying biomarkers that can aid in pinpointing those individuals who would derive the greatest benefit from therapies targeting the IL-33 pathway.
Assuntos
Asma , Imunidade Inata , Humanos , Interleucina-33/genética , Linfócitos , Asma/metabolismo , Brônquios/metabolismo , Células Epiteliais/metabolismo , Células CultivadasRESUMO
BACKGROUND: In patients with asthma, respiratory syncytial virus (RSV) infections can cause disease exacerbation by infecting the epithelial layer of the airways, inducing subsequent immune response. The type I interferon antiviral response of epithelial cells upon RSV infection is found to be reduced in asthma in most-but not all-studies. Moreover, the molecular mechanisms causing the differences in the asthmatic bronchial epithelium in response to viral infection are poorly understood. METHODS: Here, we investigated the transcriptional response to RSV infection of primary bronchial epithelial cells (pBECs) from patients with asthma (n=8) and healthy donors (n=8). The pBECs obtained from bronchial brushes were differentiated in air-liquid interface conditions and infected with RSV. After 3 days, cells were processed for single-cell RNA sequencing. RESULTS: A strong antiviral response to RSV was observed for all cell types, for all samples (p<1e-48). Most (1045) differentially regulated genes following RSV infection were found in cells transitioning to secretory cells. Goblet cells from patients with asthma showed lower expression of genes involved in the interferon response (false discovery rate <0.05), including OASL, ICAM1 and TNFAIP3. In multiciliated cells, an impairment of the signalling pathways involved in the response to RSV in asthma was observed. CONCLUSION: Our results highlight that the response to RSV infection of the bronchial epithelium in asthma and healthy airways was largely similar. However, in asthma, the response of goblet and multiciliated cells is impaired, highlighting the need for studying airway epithelial cells at high resolution in the context of asthma exacerbation.
Assuntos
Asma , Células Epiteliais , Células Caliciformes , Infecções por Vírus Respiratório Sincicial , Humanos , Asma/imunologia , Infecções por Vírus Respiratório Sincicial/imunologia , Células Caliciformes/patologia , Masculino , Feminino , Células Epiteliais/virologia , Células Epiteliais/metabolismo , Mucosa Respiratória/virologia , Mucosa Respiratória/metabolismo , Adulto , Brônquios , Pessoa de Meia-Idade , Células Cultivadas , Vírus Sinciciais Respiratórios , Cílios/patologia , Estudos de Casos e Controles , Molécula 1 de Adesão IntercelularRESUMO
INTRODUCTION: Asthma is an inflammatory airways disease encompassing multiple phenotypes and endotypes. Several studies suggested gene expression in nasal epithelium to serve as a proxy for bronchial epithelium, being a non-invasive approach to investigate lung diseases. We hypothesised that molecular differences in upper airway epithelium reflect asthma-associated differences in the lower airways and are associated with clinical expression of asthma. METHODS: We analysed nasal epithelial gene expression data from 369 patients with asthma and 58 non-asthmatic controls from the Assessment of Small Airways Involvement in Asthma study. Unsupervised hierarchical clustering was performed on asthma-associated genes. Asthma-associated gene signatures were replicated in independent cohorts with nasal and bronchial brushes data by comparing Gene Set Variation Analysis scores between asthma patients and non-asthmatic controls. RESULTS: We identified 67 higher expressed and 59 lower expressed genes in nasal epithelium from asthma patients compared with controls (false discovery rate<0.05), including CLCA1, CST1 and POSTN, genes well known to reflect asthma in bronchial airway epithelium. Hierarchical clustering revealed several molecular asthma endotypes with distinct clinical characteristics, including an endotype with higher blood and sputum eosinophils, high fractional exhaled nitric oxide, and more severe small airway dysfunction, as reflected by lower forced expiratory flow at 50%. In an independent cohort, we demonstrated that genes higher expressed in the nasal epithelium reflect asthma-associated changes in the lower airways. CONCLUSION: Our results show that the nasal epithelial gene expression profile reflects asthma-related processes in the lower airways. We suggest that nasal epithelium may be a useful non-invasive tool to identify asthma endotypes and may advance personalised management of the disease.
RESUMO
The EMBL-EBI Expression Atlas is an added value knowledge base that enables researchers to answer the question of where (tissue, organism part, developmental stage, cell type) and under which conditions (disease, treatment, gender, etc) a gene or protein of interest is expressed. Expression Atlas brings together data from >4500 expression studies from >65 different species, across different conditions and tissues. It makes these data freely available in an easy to visualise form, after expert curation to accurately represent the intended experimental design, re-analysed via standardised pipelines that rely on open-source community developed tools. Each study's metadata are annotated using ontologies. The data are re-analyzed with the aim of reproducing the original conclusions of the underlying experiments. Expression Atlas is currently divided into Bulk Expression Atlas and Single Cell Expression Atlas. Expression Atlas contains data from differential studies (microarray and bulk RNA-Seq) and baseline studies (bulk RNA-Seq and proteomics), whereas Single Cell Expression Atlas is currently dedicated to Single Cell RNA-Sequencing (scRNA-Seq) studies. The resource has been in continuous development since 2009 and it is available at https://www.ebi.ac.uk/gxa.
Assuntos
Bases de Dados Genéticas , Proteínas/genética , Proteômica , Software , Biologia Computacional , Perfilação da Expressão Gênica , Humanos , Proteínas/química , RNA-Seq , Análise de Sequência de RNA , Análise de Célula ÚnicaRESUMO
Cell type-specific differential gene expression analyses based on single-cell transcriptome datasets are sensitive to the presence of cell-free mRNA in the droplets containing single cells. This so-called ambient RNA contamination may differ between samples obtained from patients and healthy controls. Current ambient RNA correction methods were not developed specifically for single-cell differential gene expression (sc-DGE) analyses and might therefore not sufficiently correct for ambient RNA-derived signals. Here, we show that ambient RNA levels are highly sample-specific. We found that without ambient RNA correction, sc-DGE analyses erroneously identify transcripts originating from ambient RNA as cell type-specific disease-associated genes. We therefore developed a computationally lean and intuitive correction method, Fast Correction for Ambient RNA (FastCAR), optimized for sc-DGE analysis of scRNA-Seq datasets generated by droplet-based methods including the 10XGenomics Chromium platform. FastCAR uses the profile of transcripts observed in libraries that likely represent empty droplets to determine the level of ambient RNA in each individual sample, and then corrects for these ambient RNA gene expression values. FastCAR can be applied as part of the data pre-processing and QC in sc-DGE workflows comparing scRNA-Seq data in a health versus disease experimental design. We compared FastCAR with two methods previously developed to remove ambient RNA, SoupX and CellBender. All three methods identified additional genes in sc-DGE analyses that were not identified in the absence of ambient RNA correction. However, we show that FastCAR performs better at correcting gene expression values attributed to ambient RNA, resulting in a lower frequency of false-positive observations. Moreover, the use of FastCAR in a sc-DGE workflow increases the cell-type specificity of sc-DGE analyses across disease conditions.
Assuntos
Perfilação da Expressão Gênica , RNA , Humanos , RNA/metabolismo , Perfilação da Expressão Gênica/métodos , Análise de Sequência de RNA/métodos , Transcriptoma , Projetos de Pesquisa , Análise de Célula Única/métodosRESUMO
RATIONALE: Severe asthma and chronic obstructive pulmonary disease (COPD) share common pathophysiological traits such as relative corticosteroid insensitivity. We recently published three transcriptome-associated clusters (TACs) using hierarchical analysis of the sputum transcriptome in asthmatics from the Unbiased Biomarkers for the Prediction of Respiratory Disease Outcomes (U-BIOPRED) cohort comprising one Th2-high inflammatory signature (TAC1) and two Th2-low signatures (TAC2 and TAC3). OBJECTIVE: We examined whether gene expression signatures obtained in asthma can be used to identify the subgroup of patients with COPD with steroid sensitivity. METHODS: Using gene set variation analysis, we examined the distribution and enrichment scores (ES) of the 3 TACs in the transcriptome of bronchial biopsies from 46 patients who participated in the Groningen Leiden Universities Corticosteroids in Obstructive Lung Disease COPD study that received 30 months of treatment with inhaled corticosteroids (ICS) with and without an added long-acting ß-agonist (LABA). The identified signatures were then associated with longitudinal clinical variables after treatment. Differential gene expression and cellular convolution were used to define key regulated genes and cell types. MEASUREMENTS AND MAIN RESULTS: Bronchial biopsies in patients with COPD at baseline showed a wide range of expression of the 3 TAC signatures. After ICS±LABA treatment, the ES of TAC1 was significantly reduced at 30 months, but those of TAC2 and TAC3 were unaffected. A corticosteroid-sensitive TAC1 signature was developed from the TAC1 ICS-responsive genes. This signature consisted of mast cell-specific genes identified by single-cell RNA-sequencing and positively correlated with bronchial biopsy mast cell numbers following ICS±LABA. Baseline levels of gene transcription correlated with the change in RV/TLC %predicted following 30-month ICS±LABA. CONCLUSION: Sputum-derived transcriptomic signatures from an asthma cohort can be recapitulated in bronchial biopsies of patients with COPD and identified a signature of airway mast cells as a predictor of corticosteroid responsiveness.
Assuntos
Corticosteroides , Asma , Mastócitos , Doença Pulmonar Obstrutiva Crônica , Células Th2 , Humanos , Administração por Inalação , Corticosteroides/uso terapêutico , Agonistas de Receptores Adrenérgicos beta 2/uso terapêutico , Asma/tratamento farmacológico , Asma/genética , Biomarcadores , Broncodilatadores/uso terapêutico , Quimioterapia Combinada , Mastócitos/efeitos dos fármacos , Mastócitos/metabolismo , Doença Pulmonar Obstrutiva Crônica/tratamento farmacológico , Doença Pulmonar Obstrutiva Crônica/genética , Células Th2/efeitos dos fármacos , Células Th2/metabolismoRESUMO
Allergy is an ever-evolving group of disorders, which includes asthma, atopic dermatitis, rhinitis and food allergies and that currently affects over 1 billion people worldwide. This group of disorders has exploded in incidence since around the start of the 20th century, implying that genetics is not solely responsible for its development but that environmental factors have an important role. Here, Fabio Luciani and Jonathan Coquet, in their role as editors at Immunology & Cell Biology, asked nine prominent researchers in the field of allergy to define the term 'allergy', discuss the role of genetics and the environment, nominate the most important discoveries of the past decade and describe the best strategies to combat allergy at the population level going forward.
Assuntos
Asma , Dermatite Atópica , Hipersensibilidade Alimentar , HumanosRESUMO
BACKGROUND: Asthma is stratified into type 2-high and type 2-low inflammatory phenotypes. Limited success has been achieved in developing drugs that target type 2-low inflammation. Previous studies have linked IL-6 signaling to severe asthma. IL-6 cooperates with soluble-IL-6Rα to activate cell signaling in airway epithelium. OBJECTIVE: We sought to study the role of sIL-6Rα amplified IL-6 signaling in airway epithelium and to develop an IL-6+ sIL-6Rα gene signature that may be used to select asthma patients who potentially respond to anti-IL-6 therapy. METHODS: Human airway epithelial cells were stimulated with combinations of IL-6, sIL-6Rα, and inhibitors, sgp130 (Olamkicept), and anti-IL-6R (Tocilizumab), to assess effects on pathway activation, epithelial barrier integrity, and gene expression. A gene signature was generated to identify IL-6 high patients using bronchial biopsies and nasal brushes. RESULTS: Soluble-IL-6Rα amplified the activation of the IL-6 pathway, shown by the increase of STAT3 phosphorylation and stronger gene induction in airway epithelial cells compared to IL-6 alone. Olamkicept and Tocilizumab inhibited the effect of IL-6 + sIL-6Rα on gene expression. We developed an IL-6 + sIL-6Rα gene signature and observed enrichment of this signature in bronchial biopsies but not nasal brushes from asthma patients compared to healthy controls. An IL-6 + sIL-6Rα gene signature score was associated with lower levels of sputum eosinophils in asthma. CONCLUSION: sIL-6Rα amplifies IL-6 signaling in bronchial epithelial cells. Higher local airway IL-6 + sIL-6Rα signaling is observed in asthma patients with low sputum eosinophils.
Assuntos
Asma , Interleucina-6 , Humanos , Asma/diagnóstico , Asma/tratamento farmacológico , Asma/genética , Receptor gp130 de Citocina/genética , Receptor gp130 de Citocina/metabolismo , Inflamação , Interleucina-6/metabolismo , Receptores de Interleucina-6/genética , Receptores de Interleucina-6/metabolismo , Transdução de SinaisRESUMO
The field of medicine is witnessing an exponential growth of interest in artificial intelligence (AI), which enables new research questions and the analysis of larger and new types of data. Nevertheless, applications that go beyond proof of concepts and deliver clinical value remain rare, especially in the field of allergy. This narrative review provides a fundamental understanding of the core concepts of AI and critically discusses its limitations and open challenges, such as data availability and bias, along with potential directions to surmount them. We provide a conceptual framework to structure AI applications within this field and discuss forefront case examples. Most of these applications of AI and machine learning in allergy concern supervised learning and unsupervised clustering, with a strong emphasis on diagnosis and subtyping. A perspective is shared on guidelines for good AI practice to guide readers in applying it effectively and safely, along with prospects of field advancement and initiatives to increase clinical impact. We anticipate that AI can further deepen our knowledge of disease mechanisms and contribute to precision medicine in allergy.
Assuntos
Inteligência Artificial , Hipersensibilidade , Humanos , Aprendizado de Máquina , Medicina de Precisão , Hipersensibilidade/diagnóstico , Hipersensibilidade/terapiaRESUMO
BACKGROUND: Cellular diversity of the lung endothelium has not been systematically characterized in humans. We provide a reference atlas of human lung endothelial cells (ECs) to facilitate a better understanding of the phenotypic diversity and composition of cells comprising the lung endothelium. METHODS: We reprocessed human control single-cell RNA sequencing (scRNAseq) data from 6 datasets. EC populations were characterized through iterative clustering with subsequent differential expression analysis. Marker genes were validated by fluorescent microscopy and in situ hybridization. scRNAseq of primary lung ECs cultured in vitro was performed. The signaling network between different lung cell types was studied. For cross-species analysis or disease relevance, we applied the same methods to scRNAseq data obtained from mouse lungs or from human lungs with pulmonary hypertension. RESULTS: Six lung scRNAseq datasets were reanalyzed and annotated to identify >15 000 vascular EC cells from 73 individuals. Differential expression analysis of EC revealed signatures corresponding to endothelial lineage, including panendothelial, panvascular, and subpopulation-specific marker gene sets. Beyond the broad cellular categories of lymphatic, capillary, arterial, and venous ECs, we found previously indistinguishable subpopulations; among venous EC, we identified 2 previously indistinguishable populations: pulmonary-venous ECs (COL15A1neg) localized to the lung parenchyma and systemic-venous ECs (COL15A1pos) localized to the airways and the visceral pleura; among capillary ECs, we confirmed their subclassification into recently discovered aerocytes characterized by EDNRB, SOSTDC1, and TBX2 and general capillary EC. We confirmed that all 6 endothelial cell types, including the systemic-venous ECs and aerocytes, are present in mice and identified endothelial marker genes conserved in humans and mice. Ligand-receptor connectome analysis revealed important homeostatic crosstalk of EC with other lung resident cell types. scRNAseq of commercially available primary lung ECs demonstrated a loss of their native lung phenotype in culture. scRNAseq revealed that endothelial diversity is maintained in pulmonary hypertension. Our article is accompanied by an online data mining tool (www.LungEndothelialCellAtlas.com). CONCLUSIONS: Our integrated analysis provides a comprehensive and well-crafted reference atlas of ECs in the normal lung and confirms and describes in detail previously unrecognized endothelial populations across a large number of humans and mice.
Assuntos
Biomarcadores , Células Endoteliais/metabolismo , Pulmão/metabolismo , Análise de Célula Única , Capilares , Biologia Computacional/métodos , Bases de Dados Genéticas , Suscetibilidade a Doenças , Perfilação da Expressão Gênica , Sequenciamento de Nucleotídeos em Larga Escala , Humanos , Pulmão/irrigação sanguínea , Pulmão/citologia , Microcirculação , Especificidade de Órgãos , Artéria Pulmonar , Veias Pulmonares , Análise de Célula Única/métodos , TranscriptomaRESUMO
Antiviral therapies are urgently needed to treat and limit the development of severe COVID-19 disease. Ivermectin, a broad-spectrum anti-parasitic agent, has been shown to have anti-SARS-CoV-2 activity in Vero cells at a concentration of 5 µM. These limited in vitro results triggered the investigation of ivermectin as a treatment option to alleviate COVID-19 disease. However, in April 2021, the World Health Organization stated the following: "The current evidence on the use of ivermectin to treat COVID-19 patients is inconclusive." It is speculated that the in vivo concentration of ivermectin is too low to exert a strong antiviral effect. Here, we performed a head-to-head comparison of the antiviral activity of ivermectin and the structurally related, but metabolically more stable moxidectin in multiple in vitro models of SARS-CoV-2 infection, including physiologically relevant human respiratory epithelial cells. Both moxidectin and ivermectin exhibited antiviral activity in Vero E6 cells. Subsequent experiments revealed that these compounds predominantly act on the steps following virus cell entry. Surprisingly, however, in human-airway-derived cell models, both moxidectin and ivermectin failed to inhibit SARS-CoV-2 infection, even at concentrations of 10 µM. These disappointing results call for a word of caution in the interpretation of anti-SARS-CoV-2 activity of drugs solely based on their activity in Vero cells. Altogether, these findings suggest that even using a high-dose regimen of ivermectin, or switching to another drug in the same class, is unlikely to be useful for treatment of SARS-CoV-2 in humans.
Assuntos
COVID-19 , Ivermectina , Animais , Antivirais/farmacologia , Chlorocebus aethiops , Células Epiteliais , Humanos , Ivermectina/farmacologia , Macrolídeos , SARS-CoV-2 , Células Vero , Replicação ViralRESUMO
The Human Cell Atlas (HCA) consortium aims to establish an atlas of all organs in the healthy human body at single-cell resolution to increase our understanding of basic biological processes that govern development, physiology and anatomy, and to accelerate diagnosis and treatment of disease. The Lung Biological Network of the HCA aims to generate the Human Lung Cell Atlas as a reference for the cellular repertoire, molecular cell states and phenotypes, and cell-cell interactions that characterise normal lung homeostasis in healthy lung tissue. Such a reference atlas of the healthy human lung will facilitate mapping the changes in the cellular landscape in disease. The discovAIR project is one of six pilot actions for the HCA funded by the European Commission in the context of the H2020 framework programme. discovAIR aims to establish the first draft of an integrated Human Lung Cell Atlas, combining single-cell transcriptional and epigenetic profiling with spatially resolving techniques on matched tissue samples, as well as including a number of chronic and infectious diseases of the lung. The integrated Human Lung Cell Atlas will be available as a resource for the wider respiratory community, including basic and translational scientists, clinical medicine, and the private sector, as well as for patients with lung disease and the interested lay public. We anticipate that the Human Lung Cell Atlas will be the founding stone for a more detailed understanding of the pathogenesis of lung diseases, guiding the design of novel diagnostics and preventive or curative interventions.
Assuntos
Pneumopatias , Pulmão , Humanos , Proteômica , TóraxRESUMO
BACKGROUND: Despite the well-known detrimental effects of cigarette smoke (CS), little is known about the complex gene expression dynamics in the early stages after exposure. This study aims to investigate early transcriptomic responses following CS exposure of airway epithelial cells in culture and compare these to those found in human CS exposure studies. METHODS: Primary bronchial epithelial cells (PBEC) were differentiated at the air-liquid interface (ALI) and exposed to whole CS. Bulk RNA-sequencing was performed at 1 h, 4 h, and 24 h hereafter, followed by differential gene expression analysis. Results were additionally compared to data retrieved from human CS studies. RESULTS: ALI-PBEC gene expression in response to CS was most significantly changed at 4 h after exposure. Early transcriptomic changes (1 h, 4 h post CS exposure) were related to oxidative stress, xenobiotic metabolism, higher expression of immediate early genes and pro-inflammatory pathways (i.e., Nrf2, AP-1, AhR). At 24 h, ferroptosis-associated genes were significantly increased, whereas PRKN, involved in removing dysfunctional mitochondria, was downregulated. Importantly, the transcriptome dynamics of the current study mirrored in-vivo human studies of acute CS exposure, chronic smokers, and inversely mirrored smoking cessation. CONCLUSION: These findings show that early after CS exposure xenobiotic metabolism and pro-inflammatory pathways were activated, followed by activation of the ferroptosis-related cell death pathway. Moreover, significant overlap between these transcriptomic responses in the in-vitro model and human in-vivo studies was found, with an early response of ciliated cells. These results provide validation for the use of ALI-PBEC cultures to study the human lung epithelial response to inhaled toxicants.
Assuntos
Fumar Cigarros , Xenobióticos , Brônquios/metabolismo , Fumar Cigarros/efeitos adversos , Fumar Cigarros/genética , Células Epiteliais/metabolismo , Humanos , Mucosa , Nicotiana , Xenobióticos/metabolismo , Xenobióticos/farmacologiaRESUMO
BACKGROUND: Asthma is a complex disease with multiple phenotypes that may differ in disease pathobiology and treatment response. IL33 single nucleotide polymorphisms (SNPs) have been reproducibly associated with asthma. IL33 levels are elevated in sputum and bronchial biopsies of patients with asthma. The functional consequences of IL33 asthma SNPs remain unknown. OBJECTIVE: This study sought to determine whether IL33 SNPs associate with asthma-related phenotypes and with IL33 expression in lung or bronchial epithelium. This study investigated the effect of increased IL33 expression on human bronchial epithelial cell (HBEC) function. METHODS: Association between IL33 SNPs (Chr9: 5,815,786-6,657,983) and asthma phenotypes (Lifelines/DAG [Dutch Asthma GWAS]/GASP [Genetics of Asthma Severity & Phenotypes] cohorts) and between SNPs and expression (lung tissue, bronchial brushes, HBECs) was done using regression modeling. Lentiviral overexpression was used to study IL33 effects on HBECs. RESULTS: We found that 161 SNPs spanning the IL33 region associated with 1 or more asthma phenotypes after correction for multiple testing. We report a main independent signal tagged by rs992969 associating with blood eosinophil levels, asthma, and eosinophilic asthma. A second, independent signal tagged by rs4008366 presented modest association with eosinophilic asthma. Neither signal associated with FEV1, FEV1/forced vital capacity, atopy, and age of asthma onset. The 2 IL33 signals are expression quantitative loci in bronchial brushes and cultured HBECs, but not in lung tissue. IL33 overexpression in vitro resulted in reduced viability and reactive oxygen species-capturing of HBECs, without influencing epithelial cell count, metabolic activity, or barrier function. CONCLUSIONS: We identify IL33 as an epithelial susceptibility gene for eosinophilia and asthma, provide mechanistic insight, and implicate targeting of the IL33 pathway specifically in eosinophilic asthma.
Assuntos
Asma , Regulação da Expressão Gênica/imunologia , Predisposição Genética para Doença , Interleucina-33 , Polimorfismo de Nucleotídeo Único , Adulto , Asma/genética , Asma/imunologia , Feminino , Estudo de Associação Genômica Ampla , Humanos , Interleucina-33/genética , Interleucina-33/imunologia , Masculino , Pessoa de Meia-IdadeRESUMO
BACKGROUND: Differential DNA methylation associated with allergy might provide novel insights into the shared or unique etiology of asthma, rhinitis, and eczema. OBJECTIVE: We sought to identify DNA methylation profiles associated with childhood allergy. METHODS: Within the European Mechanisms of the Development of Allergy (MeDALL) consortium, we performed an epigenome-wide association study of whole blood DNA methylation by using a cross-sectional design. Allergy was defined as having symptoms from at least 1 allergic disease (asthma, rhinitis, or eczema) and positive serum-specific IgE to common aeroallergens. The discovery study included 219 case patients and 417 controls at age 4 years and 228 case patients and 593 controls at age 8 years from 3 birth cohorts, with replication analyses in 325 case patients and 1111 controls. We performed additional analyses on 21 replicated sites in 785 case patients and 2124 controls by allergic symptoms only from 8 cohorts, 3 of which were not previously included in analyses. RESULTS: We identified 80 differentially methylated CpG sites that showed a 1% to 3% methylation difference in the discovery phase, of which 21 (including 5 novel CpG sites) passed genome-wide significance after meta-analysis. All 21 CpG sites were also significantly differentially methylated with allergic symptoms and shared between asthma, rhinitis, and eczema. The 21 CpG sites mapped to relevant genes, including ACOT7, LMAN3, and CLDN23. All 21 CpG sties were differently methylated in asthma in isolated eosinophils, and 10 were replicated in respiratory epithelium. CONCLUSION: Reduced whole blood DNA methylation at 21 CpG sites was significantly associated with childhood allergy. The findings provide novel insights into the shared molecular mechanisms underlying asthma, rhinitis, and eczema.