Chronic Obstructive Pulmonary Disease Lung Tissue Research Articles

Identification of Siglec-1-negative alveolar macrophages with pro-inflammatory phenotypes in chronic obstructive pulmonary disease.

Alveolar macrophages (AMs) in patients with chronic obstructive pulmonary disease (COPD) orchestrate persistent inflammation in the airway. However, sub-populations of AMs participating in the chronic inflammation have been poorly characterized. We previously reported that Siglec-1 expression on AMs, which is important for bacteria engulfment, was decreased in COPD. Here, we show that Siglec-1-negative AMs isolated from COPD lung tissues exhibit a pro-inflammatory phenotype and is associated with poor clinical outcomes in patients with COPD. Using flow-cytometry, we segregated three subsets of AMs based on the expression of Siglec-1 and their side scattergram (SSC) and forward scattergram (FSC) properties: Siglec-1+SSChiFSChi, Siglec-1-SSChiFSChi and Siglec-1-SSCloFSClo subsets. The Siglec-1-SSCloFSClo subset number was increased in COPD. RNA-sequencing revealed upregulation of multiple pro-inflammatory signaling pathways and emphysema-associated matrix metalloproteases in the Siglec-1-SSCloFSClo subset. Gene set enrichment analysis indicated that the Siglec-1-SSCloFSClo subset adopted intermediate phenotypes between monocytes and mature alveolar macrophages. Functionally, these cells produced TNF-α, IL-6 and IL-8 at baseline, and these cytokines were significantly increased in response to viral RNA. The increase in Siglec-1-negative AMs in induced sputum is associated with future exacerbation risk and lung function decline in patients with COPD. Collectively, the novel Siglec-1-SSCloFSClo subset of AMs display pro-inflammatory properties, and their emergence in COPD airways may be associated with poor clinical outcomes.

Low dose cadmium exposure regulates miR-381–ANO1 interaction in airway epithelial cells

Chronic obstructive pulmonary disease (COPD) is the 3rd leading cause of death worldwide. Cigarette smoke which has approximately 2–3 µg of Cadmium (Cd) per cigarette contributes to the environmental exposure and development and severity of COPD. With the lack of a cadmium elimination mechanism in humans, the contribution of cadmium induced stress to lung epithelial cells remains unclear. Studies on cadmium responsive miRNAs suggest regulation of target genes with an emphasis on the critical role of miRNA–mRNA interaction for cellular homeostasis. Mir-381, the target miRNA in this study is negatively regulated by cadmium in airway epithelial cells. miR-381 is reported to also regulate ANO1 (Anoctamin 1) expression negatively and in this study low dose cadmium exposure to airway epithelial cells was observed to upregulate ANO1 mRNA expression via mir-381 inhibition. ANO1 which is a Ca2+-activated chloride channel has multiple effects on cellular functions such as proliferation, mucus hypersecretion and fibroblast differentiation in inflamed airways in chronic respiratory diseases. In vitro studies with cadmium at a high concentration range of 100–500 µM is reported to activate chloride channel, ANO1. The secretory epithelial cells are regulated by chloride channels like CFTR, ANO1 and SLC26A9. We examined “ever” smokers with COPD (n = 13) lung tissue sections compared to “never” smoker without COPD (n = 9). We found that “ever” smokers with COPD had higher ANO1 expression. Using mir-381 mimic to inhibit ANO1, we demonstrate here that ANO1 expression is significantly (p < 0.001) downregulated in COPD derived airway epithelial cells exposed to cadmium. Exposure to environmental cadmium contributes significantly to ANO1 expression.

Systematic analysis of various RNA transcripts and construction of biological regulatory networks at the post-transcriptional level for chronic obstructive pulmonary disease

BackgroundAlthough chronic inflammation, oxidative stress, airway remodeling, and protease-antiprotease imbalance have been implicated in chronic obstructive pulmonary disease (COPD), the exact pathogenesis is still obscure. Gene transcription and post-transcriptional regulation have been taken into account as key regulators of COPD occurrence and development. Identifying the hub genes and constructing biological regulatory networks at the post-transcriptional level will help extend current knowledge on COPD pathogenesis and develop potential drugs.MethodsAll lung tissues from non-smokers (n = 6), smokers without COPD (smokers, n = 7), and smokers with COPD (COPD, n = 7) were collected to detect messenger RNA (mRNA), microRNA (miRNA), circular RNA (circRNA), and long non-coding RNA (lncRNA) expression and identify the hub genes. Biological regulatory networks were constructed at the post-transcriptional level, including the RNA-binding protein (RBP)-hub gene interaction network and the competitive endogenous RNA (ceRNA) network. In addition, we assessed the composition and abundance of immune cells in COPD lung tissue and predicted potential therapeutic drugs for COPD. Finally, the hub genes were confirmed at both the RNA and protein levels.ResultsAmong the 20 participants, a total of 121169 mRNA transcripts, 1871 miRNA transcripts, 4244 circRNA transcripts, and 122130 lncRNA transcripts were detected. There were differences in the expression of 1561 mRNAs, 48 miRNAs, 33 circRNAs, and 545 lncRNAs between smokers and non-smokers, as well as 1289 mRNAs, 69 miRNAs, 32 circRNAs, and 433 lncRNAs between smokers and COPD patients. 18 hub genes were identified in COPD. TGF-β signaling and Wnt/β-catenin signaling may be involved in the development of COPD. Furthermore, the circRNA/lncRNA-miRNA-mRNA ceRNA networks and the RBP-hub gene interaction network were also constructed. Analysis of the immune cell infiltration level revealed that M2 macrophages and activated NK cells were increased in COPD lung tissues. Finally, we identified that the ITK inhibitor and oxybutynin chloride may be effective in treating COPD.ConclusionsWe identified several novel hub genes involved in COPD pathogenesis. TGF-β signaling and Wnt/β-catenin signaling were the most dysregulated pathways in COPD patients. Our study constructed post-transcriptional biological regulatory networks and predicted small-molecule drugs for the treatment of COPD, which enhanced the existing understanding of COPD pathogenesis and suggested an innovative direction for the therapeutic intervention of the disease.

Identification and Analysis of Necroptosis-Related Genes in COPD by Bioinformatics and Experimental Verification

Chronic obstructive pulmonary disease (COPD) is a heterogeneous and complex progressive inflammatory disease. Necroptosis is a newly identified type of programmed cell death. However, the role of necroptosis in COPD is unclear. This study aimed to identify necroptosis-related genes in COPD and explore the roles of necroptosis and immune infiltration through bioinformatics. The analysis identified 49 differentially expressed necroptosis-related genes that were primarily engaged in inflammatory immune response pathways. The infiltration of CD8+ T cells and M2 macrophages in COPD lung tissue was relatively reduced, whereas that of M0 macrophages was increased. We identified 10 necroptosis-related hub genes significantly associated with infiltrated immune cells. Furthermore, 7 hub genes, CASP8, IL1B, RIPK1, MLKL, XIAP, TNFRSF1A, and CFLAR, were validated using an external dataset and experimental mice. CFLAR was considered to have the best COPD-diagnosing capability. TF and miRNA interactions with common hub genes were identified. Several related potentially therapeutic molecules for COPD were also identified. The present findings suggest that necroptosis occurs in COPD pathogenesis and is correlated with immune cell infiltration, which indicates that necroptosis may participate in the development of COPD by interacting with the immune response.

Long noncoding RNA HOTAIR facilitates pulmonary vascular endothelial cell apoptosis via DNMT1 mediated hypermethylation of Bcl-2 promoter in COPD

BackgroundTo study the regulatory effect of Long non-coding RNA (LncRNA) HOX transcript antisense RNA (HOTAIR) on pulmonary vascular endothelial cell (HPVEC) apoptosis and determine whether the HOTAIR facilitate HPVEC apoptosis via DNMT1 mediated hypermethylation of Bcl-2 promoter in chronic obstructive pulmonary disease (COPD).MethodsLncRNA array was used to measure the differentially expressed lncRNAs in COPD and non-COPD lung tissues. Expression of HOTAIR in COPD patient lungs and cigarette smoke extract (CSE)-induced HPVEC was assessed by qRT-PCR. The location of HOTAIR was determined in COPD patient lungs and HPVEC by RNA in situ hybridization (RNA-ISH). The emphysema mouse model and HOTAIR knockdown mice were each established by inhaling cigarette smoke or intratracheal lentiviral vectors instillation. The dysregulation of DNA methyltransferase enzyme 1 (DNMT1), B-cell lymphoma-2 (Bcl-2), Bcl-2-associated X protein (Bax) and Cleaved-caspase 3 protein expression were detected by Western blotting. HOTAIR, DNMT1, Bcl-2 and Bax mRNA expression were measured by quantitative real-time polymerase chain reaction. TUNEL (terminal deoxynucleotidyl transferase dUTP nick end labeling) assays were used to assess apoptotic ratio in mice and CSE-induced HPVEC. Methylation-specific PCR (MSP) assay was conducted to observe the alterations in the methylation of the Bcl-2 promoter in specimens. RNA pull-down assay was used for analysis of the correlation between HOTAIR and DNMT1.ResultsThe expression levels of the HOTAIR were up-regulated in COPD patient lungs and CSE-induced HPVEC. HPVEC apoptosis with down-regulated Bcl-2 expression, increased promoter methylation, DNMT1, Bax and Cleaved-caspase 3 expression was found in emphysema mouse model and CSE-induced HPVEC. Knockdown HOTAIR can attenuate cell apoptosis and emphysema via DNMT1 mediated hypermethylation of Bcl-2 promoter in mice. In vitro, HOTAIR can aggravate the apoptosis of CSE-exposed HPVEC. DNMT1 was a target of HOTAIR and had a positive correlation with HOTAIR.ConclusionHOTAIR facilitates HPVEC apoptosis via DNMT1 mediated hypermethylation of Bcl-2 promoter in COPD, and attenuating the expression of HOTAIR may be a new therapy to prevent COPD.

Hypoxic conditions promote a proliferative, poorly differentiated phenotype in COPD lung tissue progenitor cells in vitro

PurposeChronic obstructive pulmonary disease (COPD) patients experience hypoxemia and lung tissue hypoxia, causing vasoconstriction, and at its most severe Cor pulmonale. However, minimal attention has been given to the effects of hypoxia at the cellular level. We hypothesize that a persistent progenitor cell population undergoes an aberrant differentiation process, influenced by changes in oxygen.MethodsDistal lung progenitor cells from two emphysematous donors were cultured in 21% and 2% oxygen. Proliferation was determined on collagen-coated plastic and in 3T3-J2 co-culture. Epithelial (E-cadherin, pan-cytokeratin) and progenitor (TP63, cytokeratin 5) marker expressions were examined. Cells were differentiated at air–liquid interface, and ciliated, mucus-producing, and club cell populations identified by immunofluorescence. MUC5AC, MUC5B, CC10, and TP63 expression were determined using qRT-PCR, mucin5AC, and mucin5B protein levels by ELISA, and secreted mucin by periodic acid biotin hydrazide assay.ResultsCells were positive for epithelial and progenitor markers at isolation and passage 5. Passage 5 cells in hypoxia increased the proportion of TP63 by 10% from 51.6 ± 1.2% to 62.6 ± 2.3% (p ≤ 0.01). Proliferative capacity was greater on 3T3J2 cells and in 2% oxygen, supporting the emergence of a proliferation unrestricted population with limited differentiation capacity. Differentiation resulted in βIV tubulin positive-ciliated cells, mucin5AC, mucin5B, and CC10 positive secretory cells. Epithelial barrier formation was reduced (p ≤ 0.0001) in hypoxia-expanded cells. qRT-PCR showed higher mucin expression in 2% cells, significantly so with MUC5B (p ≤ 0.05). Although overall mucin5AC and mucin5B content was greater in 21% cells, normalization of secreted mucin to DNA showed a trend for increased mucin by low oxygen cells.ConclusionsThese results demonstrate that hypoxia promotes a proliferative phenotype while affecting subsequent progenitor cell differentiation capacity. Furthermore, the retained differentiation potential becomes skewed to a more secretory phenotype, demonstrating that hypoxia may be contributing to disease symptoms and severity in COPD patients.

MiR-378 associated with proliferation, migration and apoptosis properties in A549 cells and targeted NPNT in COPD.

BackgroundmicroRNAs contribute to the development and progression of chronic obstructive pulmonary disease (COPD). However, the underlying molecular mechanisms are largely unclear. The goal of this study was to investigate the roles of miR-378 in alveolar epithelial type II cells and identify molecular mechanisms which contribute to the pathogenesis of COPD.Materials and methodsHuman alveolar epithelial (A549) cells were cultured in Dulbecco’s Modified Eagle Medium. Cell proliferation was studied by using a cell counting kit-8 (CCK-8) and colony formation assays. Cell apoptosis and cell cycle were analyzed by flow cytometry and wound healing and Transwell were used to analyze the cell migration and. We performed bioinformatics analysis including target gene prediction, gene ontology (GO), Kyoto Encyclopedia of Genes and Genome (KEGG) pathway enrichment and construction of protein-protein interaction (PPI) network. The expression of miR-378 and NPNT from publically available expression microarray of COPD lung tissues was analyzed.ResultsOverexpression of miR-378 significantly increases cell proliferation, migration, and suppress apoptosis. GO analysis demonstrated that the miR-378 involved in transcription, vascular endothelial growth factor receptor signaling pathway, phosphatidylinositol 3-kinase signaling, cell migration, blood coagulation, cell shape, protein stabilization and phosphorylation. Pathway enrichment showed that the 1,629 target genes of miR-378 were associated with mTOR, ErbB, TGF-β, MAPK, and FoxO signaling pathways. Notably, miR-378 directly targets Nephronectin in A549 cells, and miR-378 was upregulated while NPNT was downregulated in COPD lung tissue samples.ConclusionsThese findings suggest that miR-378 can regulate the proliferation, migration, and apoptosis of A549 cells and target NPNT. miR-378 increased in COPD lung tissues while NPNT decreased, and might prove a potential target for novel drug therapy.

Loss of core-fucosylation of SPARC impairs collagen binding and contributes to COPD

Chronic obstructive pulmonary disease (COPD) is a progressive lung disease with high morbidity and mortality worldwide. Although several mechanisms to account for deleterious immune effects were proposed, molecular description for the underlying alveolar structural alterations for COPD is lacking. Here, silencing of α1,6-fucosyltransferase (Fut8), the enzyme for core-fucosylation and highly expressed in lung stem cells, resulted in alveolar structural changes in lung organoids, recapitulating COPD. Site-specific mass spectrometry analysis demonstrated that the secreted protein acidic and rich in cysteine (SPARC), which binds collagen, contains a core-fucosylation site in its VCSNDNcfK glycopeptide. Biacore assay showed markedly reduced collagen binding of SPARC lacking core fucosylation. Molecular dynamics analysis revealed that core fucosylation of SPARC-induced dynamic conformational changes in its N-glycan, allowing terminal galactose and N-acetylglucosamine to interact with K150, P261 and H264 residues, thereby promoting collagen binding. Site-specific mutagenesis of these residues also resulted in low affinity for collagen binding. Moreover, loss of collagen and decline of core fucosylation were observed in COPD lung tissues. These findings provide a new mechanistic insight into the role of core fucosylation of SPARC in cell–matrix communication and contribution to the abnormal alveolar structures in COPD.

Identification of Ferroptosis-Related Hub Genes and Their Association with Immune Infiltration in Chronic Obstructive Pulmonary Disease by Bioinformatics Analysis.

PurposeFerroptosis and immune infiltration are involved in the pathogenesis of chronic obstructive pulmonary disease (COPD). We aim to identify ferroptosis-related hub genes and analyze their association with immune infiltration in COPD through bioinformatics methods.Materials and MethodsThe mRNA microarray data of GSE38974 were downloaded from Gene Expression Omnibus to obtain differentially expressed genes (DEGs). The DEGs were intersected with ferroptosis-related genes (FRGs) from FerrDb to obtain differentially expressed FRGs. GO and KEGG enrichment and protein–protein interaction (PPI) analyses of differentially expressed FRGs were conducted in R software and STRING database. The key module and hub genes were screened by Cytoscape software. MiRNAs, transcription factors and signal molecules were predicted in miRNet and NetworkAnalyst. The disease correlation in the Comparative Toxicomics Database (CTD) and the receiver operating characteristic (ROC) curves of hub genes were analyzed. Immune infiltration was evaluated by CIBERSORT algorithm. Spearman correlation analyses were conducted between hub genes and differentially infiltrated immune cells.ResultsFifteen differentially expressed FRGs were identified, which were enriched in some terms involving airway inflammatory responses and structural remodeling. Five hub genes were selected including HIF1A, IL6, PTGS2, CDKN1A and ATM. Inference scores in CTD indicated their association with COPD. Two miRNAs, five transcription factors and one signal molecule were predicted. The combination of hub genes could be a fine diagnostic indicator of COPD (AUC: 0.981, CI: 0.940-1.000). Immune infiltration evaluation showed that monocytes and M0 macrophages were upregulated in COPD lung tissues, while CD8 T cells, activated NK cells, M2 macrophages, resting dendritic cells and resting mast cells were downregulated. The hub genes were significantly associated with differentially infiltrated immune cells.ConclusionWe identified five ferroptosis-related hub genes (HIF1A, IL6, PTGS2, CDKN1A and ATM) in COPD, and found that they may influence the pathogenesis of COPD by regulating ferroptosis and thus affecting infiltrating immune cells.

CD19 and POU2AF1 are Potential Immune-Related Biomarkers Involved in the Emphysema of COPD: On Multiple Microarray Analysis.

PurposeEmphysema is the main cause of the progression of chronic obstructive pulmonary disease (COPD). This study aimed to identify the key genes involved in COPD-related emphysema.Patients and MethodsGSE76925 was downloaded from Gene Expression Omnibus database. Protein–protein interaction networks of differentially expressed genes (DEGs) between control and COPD groups were constructed to identify hub genes using Cytoscape. Diagnostic performance of hub genes was evaluated using receiver operating characteristic analysis. Correlation analysis was performed to identify the key genes by analyzing the relationship between the hub genes and lung function and computed tomography (CT) indexes of emphysema. COPD patients were then divided into two groups based on the median expression of key genes and DEGs between these two groups were identified. Enrichment analysis of DEGs and correlation analysis between key genes and the infiltration of the immune cells were also analyzed. Finally, the role of key genes was evaluated in a lung tissues dataset (GSE47460) and a blood dataset (GSE76705). Additionally, the expression of key genes was validated by quantitative real-time polymerase chain reaction and immunohistochemistry.ResultsCD19 and POU2AF1 had diagnostic efficacy for COPD and were significantly correlated with lung function and CT indexes of emphysema. Enrichment and immune analyses revealed that CD19 and POU2AF1 were correlated with the B cells in COPD. These results were consistent in GSE47460. The expression of CD19 and POU2AF1 in blood was the opposite of that in lung tissues, and CD19 and POU2AF1 were both significantly upregulated in COPD lung tissues at both the mRNA and protein levels.ConclusionCD19 and POU2AF1 may serve as key regulators of emphysema and contribute to the progression of COPD by regulating the B-cell immunology. Targeting B cells may be a promising strategy for treating COPD.

Increased LHX9 expression in alveolar epithelial type 2cells of patients with chronic obstructive pulmonary disease.

Alveolar epithelial type 2 (AT2) cells serve as stem cells in alveolar epithelium and are assumed to lose their stem cell function in the lungs of chronic obstructive pulmonary disease (COPD). Although we previously reported that LHX9 mRNA expression was up-regulated in AT2 cells of COPD lung tissues, it is yet to be elucidated how LHX9 is associated with the vulnerability of AT2 cells in COPD. AT2 cells were isolated from lung tissues of 10 non-COPD subjects and 11 COPD patients. LHX9 mRNA expression was determined by quantitative RT-PCR. To identify up-stream molecules, an alveolar epithelial cell line A549 was exposed to pro-inflammatory cytokines invitro. siRNA-mediated Lhx9 knockdown was performed to determine how Lhx9 affected the cellular viability and the cell-division cycle. LHX9 mRNA expression was increased in AT2 cells from COPD lung tissues, compared to those from non-COPD tissues. The airflow obstruction was independently correlated with the increase in LHX9 expression. Among several pro-inflammatory cytokines, interferon-γ was a strong inducer of LHX9 expression in A549cells. Lhx9 was involved in the increased susceptibility to serum starvation-induced death of A549cells. Our data suggest that IFN-γ predominantly increases the LHX9 expression which enhances the susceptibility to cell death. Considering the independent association of the increased LHX9 expression in AT2 cells with airflow obstruction, the IFN-γ-Lhx9 axis might contribute to the vulnerability of AT2 cells in the lungs of COPD patients.

Enhanced carbonic anhydrase expression with calcification and fibrosis in bronchial cartilage during COPD

Pulmonary cartilage plays a crucial structural role determining the physiologic airway compressibility and distensibility, necessary for proper mechanical function. This functionality deteriorates with aging due to increased stiffness of both airway muscle and cartilage, as well as, decreased renewal capacity. Altered airway remodeling has been suggested as a pathogenic driver of chronic obstructive pulmonary disease (COPD) through mechanisms still incompletely understood. Using paraffin-embedded lung tissue sections from archived autopsy material from COPD with non-COPD age matched controls a histopathologic analysis focused on inflammation, fibrosis and calcification was performed with special stains (Masson’s trichrome and Von Kossa) and immunohistochemistry for carbonic anhydrase IV (CA IV) and Ki-67. COPD lung tissues showed increased peribronchial inflammation compared to the non-COPD. Coarse amphophilic crystalline deposits in bronchial cartilage were more frequently observed in COPD sections, which were compatible with early dystrophic calcification of the extracellular matrix and chondrocytes. Moreover, Von Kossa staining revealed a significant calcium deposition in the cartilages from COPD in comparison to the controls. Interestingly, Ki-67 immunostains demonstrated a higher overall proliferative rate, including epithelial cells, in COPD. Furthermore, Masson’s trichrome staining revealed relatively increased peribronchial collagen deposition associated with a fibrotic stromal response, which may be secondary to the inflammatory milieu in COPD. To further characterize the tissue microenvironment associated with dystrophic calcification, immunohistochemistry for CA IV was used, revealing significantly increased expression in chondrocytes and peribronchial tissue in COPD. Our findings demonstrate that dystrophic calcification of the extracellular matrix and chondrocytes can be linked to CA IV expression in COPD and suggest that pH changes in pulmonary tissue associated with inflammation and calcification may play an active role in COPD.

MicroRNA miR-24-3p reduces DNA damage responses, apoptosis, and susceptibility to chronic obstructive pulmonary disease

The pathogenesis of chronic obstructive pulmonary disease (COPD) involves aberrant responses to cellular stress caused by chronic cigarette smoke (CS) exposure. However, not all smokers develop COPD and the critical mechanisms that regulate cellular stress responses to increase COPD susceptibility are not understood. Because microRNAs are well-known regulators of cellular stress responses, we evaluated microRNA expression arrays performed on distal parenchymal lung tissue samples from 172 subjects with and without COPD. We identified miR-24-3p as the microRNA that best correlated with radiographic emphysema and validated this finding in multiple cohorts. In a CS exposure mouse model, inhibition of miR-24-3p increased susceptibility to apoptosis, including alveolar type II epithelial cell apoptosis, and emphysema severity. In lung epithelial cells, miR-24-3p suppressed apoptosis through the BH3-only protein BIM and suppressed homology-directed DNA repair and the DNA repair protein BRCA1. Finally, we found BIM and BRCA1 were increased in COPD lung tissue, and BIM and BRCA1 expression inversely correlated with miR-24-3p. We concluded that miR-24-3p, a regulator of the cellular response to DNA damage, is decreased in COPD, and decreased miR-24-3p increases susceptibility to emphysema through increased BIM and apoptosis.

Role of long non-coding RNA MALAT1 in chronic obstructive pulmonary disease.

Chronic obstructive pulmonary disease (COPD) is a pathological inflammatory condition of the lungs that is associated with high rates of mortality. Although long non-coding RNAs (lncRNAs) serve a role in lung diseases, their functions in COPD pathogenesis are relatively unknown. The present study aimed to assess the role of differentially expressed lncRNAs in COPD. Expression profile analysis of six lncRNAs in age-matched COPD and non-COPD tissues were conducted. Among the six tested lncRNAs, metastasis-associated in lung adenocarcinoma transcript 1 (MALAT1) was the most consistently overexpressed in COPD lung tissue specimens. To model COPD in vitro, human lung fibroblasts were treated with transforming growth factor-β (TGF-β) and MALAT1 was knocked down by small interfering RNA. This promoted cell viability and concurrently inhibited the expression of mesenchymal proteins, fibronectin and α-smooth muscle actin. In COPD, cell senescence is linked to the activation of mammalian target of rapamycin complex 1 (mTORC1). Upon gene silencing of MALAT1 in non-TGF-β-treated cells, cells demonstrated constitutive activation of mTORC1, which was assessed by the protein expression levels of mTORC1 substrate S6 kinase (S6K1). By contrast, upon MALAT1 silencing in the TGF-β-treated cells, mTORC1 activation was not suppressed, despite the mesenchymal cell markers protein expression levels being downregulated. Thus, lncRNA MALAT1 may represent a potent biomarker in COPD patients and may act as a target for both diagnostic and therapeutic purposes.

Integrated proteogenomic approach identifying a protein signature of COPD and a new splice variant of SORBS1

Translation of genomic alterations to protein changes in chronic obstructive pulmonary disease (COPD) is largely unexplored. Using integrated proteomic and RNA sequencing analysis of COPD and control lung tissues, we...

The modulatory effects of the PDE4 inhibitors CHF6001 and roflumilast in alveolar macrophages and lung tissue from COPD patients

BackgroundWe compared the anti-inflammatory effects of phosphodiesterase type 4 (PDE4) inhibitor roflumilast with CHF6001, a novel PDE4 inhibitor designed for inhaled administration, using human alveolar macrophages (AM) and lung tissue explants models. MethodsAM from 13 chronic obstructive pulmonary disease (COPD) patients and 10 smoking controls and lung tissue from 7 COPD patients were stimulated with LPS following preincubation with roflumilast (0.000001–10 µM), CHF6001 (0.000001–0.1 µM), or vehicle. After 24 h, supernatants were analysed for cytokines by ELISA. The effects of both compounds on the phosphorylation and cellular localisation of cAMP response element binding protein (CREB) were assessed by immunofluorescence and Western blot analysis. Extracted RNA was used for quantitative PCR analysis of PDE4 A, B and D mRNA. ResultsPDE4 A, B and D expression were increased in alveolar macrophages and lung tissue of COPD patients compared to controls. Roflumilast and CHF6001 significantly reduced TNF-α production in AM and lung tissue. CHF6001 was more potent than roflumilast with lower EC50s of 0.02, 0.01 and 0.31 nM compared to 0.87, 0.47 and 10.8 nM in respective samples. PDE4 inhibition also inhibited secretion of the chemokines CCL2 and CCL4 from macrophages. Both compounds increased nuclear levels of phosphorylated CREB. ConclusionPDE4 inhibitors caused a robust anti-inflammatory effect on TNF-α production from COPD AM, with inhibition of selective chemokines also observed. CHF6001 caused more potent inhibition of TNF-α production from COPD AM and lung tissue compared to roflumilast.

Exploring the cross-phenotype network region of disease modules reveals concordant and discordant pathways between chronic obstructive pulmonary disease and idiopathic pulmonary fibrosis.

Chronic obstructive pulmonary disease (COPD) and idiopathic pulmonary fibrosis (IPF) are two pathologically distinct chronic lung diseases that are associated with cigarette smoking. Genetic studies have identified shared loci for COPD and IPF, including several loci with opposite directions of effect. The existence of additional shared genetic loci, as well as potential shared pathobiological mechanisms between the two diseases at the molecular level, remains to be explored. Taking a network-based approach, we built disease modules for COPD and IPF using genome-wide association studies-implicated genes. The two disease modules displayed strong disease signals in an independent gene expression data set of COPD and IPF lung tissue and showed statistically significant overlap and network proximity, sharing 19 genes, including ARHGAP12 and BCHE. To uncover pathways at the intersection of COPD and IPF, we developed a metric, NetPathScore, which prioritizes the pathways of a disease by their network overlap with another disease. Applying NetPathScore to the COPD and IPF disease modules enabled the determination of concordant and discordant pathways between these diseases. Concordant pathways between COPD and IPF included extracellular matrix remodeling, Mitogen-activated protein kinase (MAPK) signaling and ALK pathways, whereas discordant pathways included advanced glycosylation end product receptor signaling and telomere maintenance and extension pathways. Overall, our findings reveal shared molecular interaction regions between COPD and IPF and shed light on the congruent and incongruent biological processes lying at the intersection of these two complex diseases.

A large lung gene expression study identifying IL1B as a novel player in airway inflammation in COPD airway epithelial cells.

Chronic obstructive pulmonary disease (COPD) is a chronic and progressive lung disease characterized by a mixture of small airway disease and lung tissue parenchymal destruction. Abnormal inflammatory responses to cigarette smoking and other noxious particles are generally thought to be responsible for causing of COPD. Since airway inflammation is a key factor in COPD progress, it is crucial to unravel its underlying molecular mechanisms. Unbiased analysis of genome-wide gene expression profiles in lung small airway epithelial cells provides a powerful tool to investigate this. Gene expression data of GSE611906, GSE20257, GSE8545 were downloaded from GEO database. All 288 lung small airway samples in these cohorts, including donors with (n = 61) and without (n = 227) COPD, were chosen for differential gene expression analysis. The gene ontology (GO) function, Kyoto Encyclopedia of Genes and Genomes pathway (KEGG) enrichment analyses, gene co-expression network analysis (WGCNA) and protein-protein interaction (PPI) network analysis were performed. Subsequently, the analyses of IL1B expression level, the Pearson correlation between IL1B and several COPD biomarkers were performed using other cohorts to validate our main findings. With a change ≥ twofold and P value < 0.05 cutoff, we found 38 genes were up-regulated and 114 genes were down-regulated in patients with COPD compared with health controls, while using cutoff fold change 1.5 and P value < 0.05, there were 318 genes up-regulated and 333 genes down-regulated. Among the most up-regulated genes were IL1B, CCL2, CCL23, and CXCL14, all implicated in inflammation triggering. GO, KEGG and WGCNA analysis all disclosed IL1B was highly correlated to COPD disease trait. The expression profile of IL1B was further validated using independent cohorts from COPD airway epithelium, lung tissue, sputum, and blood. We demonstrated higher IL1B gene expression in COPD small airway epithelial cells, but not in COPD lung tissue, sputum, and blood. Strong co-expression of IL1B with COPD biomarkers, such as DUOX2, MMP12, CCL2, and CXCL14, were validated in silico analysis. Finally, PPI network analysis using enriched data showed IL1B, CCL2, CCL7 and BMP7 were in the same hub node with high degrees. We identified IL1B was significantly up-regulated in COPD small airway epithelial cells and propose IL1B as a novel player in airway inflammation in COPD.

The lung tissue microbiota of mild and moderate chronic obstructive pulmonary disease

BackgroundOral taxa are often found in the chronic obstructive pulmonary disease (COPD) lung microbiota, but it is not clear if this is due to a physiologic process such as aspiration or experimental contamination at the time of specimen collection.MethodsMicrobiota samples were obtained from nine subjects with mild or moderate COPD by swabbing lung tissue and upper airway sites during lung lobectomy. Lung specimens were not contaminated with upper airway taxa since they were obtained surgically. The microbiota were analyzed with 16S rRNA gene qPCR and 16S rRNA gene hypervariable region 3 (V3) sequencing. Data analyses were performed using QIIME, SourceTracker, and R.ResultsStreptococcus was the most common genus in the oral, bronchial, and lung tissue samples, and multiple other taxa were present in both the upper and lower airways. Each subject’s own bronchial and lung tissue microbiota were more similar to each other than were the bronchial and lung tissue microbiota of two different subjects (permutation test, p = 0.0139), indicating more within-subject similarity than between-subject similarity at these two lung sites. Principal coordinate analysis of all subject samples revealed clustering by anatomic sampling site (PERMANOVA, p = 0.001), but not by subject. SourceTracker analysis found that the sources of the lung tissue microbiota were 21.1% (mean) oral microbiota, 8.7% nasal microbiota, and 70.1% unknown. An analysis using the neutral theory of community ecology revealed that the lung tissue microbiota closely reflects the bronchial, oral, and nasal microbiota (immigration parameter estimates 0.69, 0.62, and 0.74, respectively), with some evidence of ecologic drift occurring in the lung tissue.ConclusionThis is the first study to evaluate the mild-moderate COPD lung tissue microbiota without potential for upper airway contamination of the lung samples. In our small study of subjects with COPD, we found oral and nasal bacteria in the lung tissue microbiota, confirming that aspiration is a source of the COPD lung microbiota.

The Lung Tissue Microbiota of Mild and Moderate Chronic Obstructive Pulmonary Disease

Oral taxa are often found in the chronic obstructive pulmonary disease (COPD) lung microbiota, but it is not clear if this is due to a physiologic process such as aspiration or experimental contamination at the time of specimen collection. Microbiota samples were obtained from nine subjects with mild or moderate COPD by swabbing lung tissue and upper airway sites during lung lobectomy. Lung specimens were not contaminated with upper airway taxa since they were obtained surgically. The microbiota were analyzed with 16S rRNA gene qPCR and 16S rRNA gene hypervariable region 3 (V3) sequencing. Data analyses were performed using QIIME, SourceTracker, and R. Streptococcus was the most common genus in the oral, bronchial, and lung tissue samples, and multiple other taxa were present in both the upper and lower airways. Each subject’s own bronchial and lung tissue microbiota were more similar to each other than were the bronchial and lung tissue microbiota of two different subjects (permutation test, p = 0.0139), indicating more within-subject similarity than between-subject similarity at these two lung sites. Principal coordinate analysis of all subject samples revealed clustering by anatomic sampling site (PERMANOVA, p = 0.001), but not by subject. SourceTracker analysis found that the sources of the lung tissue microbiota were 21.1% (mean) oral microbiota, 8.7% nasal microbiota, and 70.1% unknown. An analysis using the neutral theory of community ecology revealed that the lung tissue microbiota closely reflects the bronchial, oral, and nasal microbiota (immigration parameter estimates 0.69, 0.62, and 0.74, respectively), with some evidence of ecologic drift occurring in the lung tissue. This is the first study to evaluate the mild-moderate COPD lung tissue microbiota without potential for upper airway contamination of the lung samples. In our small study of subjects with COPD, we found oral and nasal bacteria in the lung tissue microbiota, confirming that aspiration is a source of the COPD lung microbiota.