Intra-tracheal delivery of AAV6 vectors results in sustained transduction in murine lungs without genomic integration

Human mucin (MUC) 5B gene expression in human airway epithelium was studied in both tissue sections and cultures of tracheobronchial epithelial (TBE) cells. In situ hybridization demonstrated that MUC5B message was expressed mainly in the mucous cells of submucosal glands of normal human airway tissues. Nevertheless, an elevated MUC5B message level could be seen in surface goblet cells from patients with airway diseases and inflammation. Regardless of the airway tissue sources, MUC5B message was regulated by all-trans-retinoic acid (RA) and culture conditions in both primary and passage-1 cultures of TBE cells. MUC5B message, to a lesser extent, was also found in the immortalized epithelial cell line HBE1, but not in BEAS-2B cells. To elucidate the molecular mechanism of MUC5B gene expression, a genomic clone was obtained and sequenced for the amino terminal and the 5'-flanking region of MUC5B gene. A luciferase reporter construct containing 4,169 base pairs of the 5'-flanking region of MUC5B gene demonstrated a cell type-specific basal promoter activity in transfection studies. Both RA and the air-liquid interface culture condition further enhanced this promoter activity. These results suggest that the 5'-flanking region of MUC5B gene contains cis-elements that are potentially involved in the regulation of MUC5B gene expression.

Eotaxin is an eosinophil-specific chemoattractant originally identified in bronchoalveolar lavage fluid after allergen challenge of sensitized guinea-pigs. We have determined and quantified for the first time the cellular sources of guinea-pig lung eotaxin and localized gene expression in structural cells of large and small airways and in alveolar macrophages. We used anti-guinea-pig eotaxin monoclonal and polyclonal antibodies and a complementary ribonucleic acid (cRNA) probe to detect eotaxin protein and cytoplasmic messenger ribonucleic acid (mRNA) transcripts by the techniques of immunohistochemistry and in situ hybridization in: 1) naive; 2) ovalbumin-sensitized/ saline-exposed; and 3) ovalbumin-sensitized/ovalbumin-challenged animals (n = 5 for each group). Compared with the naive animals, there was a fivefold increase of eotaxin protein and a 25 fold upregulation of eotaxin gene expression in the airway epithelium 3 h after ovalbumin challenge of sensitized animals (p < 0.001). The average percentages of alveolar macrophages staining for eotaxin protein and mRNA in the naive animals were approximately 30 and 10% respectively: both increased significantly in the sensitized/ovalbumin-challenged animals to 78 and 57%, respectively (p < 0.0001). Compared with the naive animals, the procedure of sensitization and saline exposure significantly increased eotaxin gene expression in both bronchial epithelium and alveolar macrophages (p < 0.01): the upregulation at these two sites showed a strong positive association (rcorr = 0.95; p < 0.0001). The results indicate that there are multiple cellular sources of guinea-pig lung-derived eotaxin, including bronchial and bronchiolar epithelial cells, airway smooth muscle, bronchial vascular endothelium, and chondrocytes and alveolar macrophages, and that there are relatively rapid and marked increases of eotaxin protein and gene expression in airway epithelium and alveolar macrophages following allergen challenge.

Cis- and trans-eQTM analysis reveals novel epigenetic and transcriptomic immune markers of atopic asthma in airway epithelium

Expression Quantitative Trait Methylation Analysis Reveals Methylomic Associations With Gene Expression in Childhood Asthma

Using microarray gene expression analysis, we first observed a profound elevation of human beta-defensin-2 (hBD-2) message in IL-17-treated primary human airway epithelial cells. Further comparison of this stimulation with a panel of cytokines (IL-1alpha, 1beta, 2-13, and 15-18; IFN-gamma; GM-CSF; and TNF-alpha) demonstrated that IL-17 was the most potent cytokine to induce hBD-2 message (>75-fold). IL-17-induced stimulation of hBD-2 was time and dose dependent, and this stimulation also occurred at the protein level. Further studies demonstrated that hBD-2 stimulation was attenuated by IL-17R-specific Ab, but not by IL-1R antagonist or the neutralizing anti-IL-6 Ab. This suggests an IL-17R-mediated signaling pathway rather than an IL-17-induced IL-1alphabeta and/or IL-6 autocrine/paracrine loop. hBD-2 stimulation was sensitive to the inhibition of the JAK pathway, and to the inhibitors that affect NF-kappaB translocation and the DNA-binding activity of its p65 NF-kappaB subunit. Transient transfection of airway epithelial cells with an hBD-2 promoter-luciferase reporter gene expression construct demonstrated that IL-17 stimulated promoter-reporter gene activity, suggesting a transcriptional mechanism for hBD-2 induction. These results support an IL-17R-mediated signaling pathway involving JAK and NF-kappaB in the transcriptional stimulation of hBD-2 gene expression in airway epithelium. Because IL-17 has been identified in a number of airway diseases, especially diseases related to microbial infection, these findings provide a new insight into how IL-17 may play an important link between innate and adaptive immunity, thereby combating infection locally within the airway epithelium.

Background: Previous GWAS studies have detected genetic variants that affect the risk to develop COPD. For most of these genetic variants it is not clear yet how they contribute to COPD and which cells they affect. We hypothesized that part of these genetic variants contribute to COPD susceptibility by affecting gene expression in airway epithelium by acting as expression QTLs (eQTLs). Methods: The gene expression (Affymetrix Hugene arrays) and genotype information (overlap between the Illumina OmniExpress Exome and HumanCytoSNP-12) was obtained from 150 bronchial brushings from 140 healthy individuals and 10 with mild COPD. 26 SNPs were selected from four previous GWAS studies focusing on FEV1 or FEV1/FVC. Each SNP was then tested for their effect on gene expression using a linear model, correcting for: age, smoking, gender and PCs. To predict target genes, we examined domains that had long-range three-dimensional chromatin interactions surrounding each tested SNP. This was conducted Hi-C data from the lung cell line A549. Results: Of the 26 previous GWAS hits for FEV1 and FEV1/FVC, 15 were present or had a proxy SNP (r2>0.8). Hi-C analysis identified 5-43 genes which were in position of direct interaction with each tested SNP. EQTL analysis of the identified genes identified one SNP to be associated with three cis-eQTLs (FDR Conclusions: This study for the first time investigates GWAS SNP associated with FEV1/FVC in an epithelial specific eQTL database, providing evidence that lung function SNPs are associated with eQTLs in airway epithelium.

Chronic obstructive pulmonary disease (COPD) is one of the most socially significant respiratory diseases. The development and progression of COPD is mainly associated with smoking, which leads to chronic inflammation of the respiratory tract, including both cellular and humoral immunological processes. Contribution in immune defense of the lungs of ATP-transporters (ABC) is of great interest. Three lipid transporters ABCA1, ABCG1 and ABCA3 are expressed in lung cells. The first two contribute to reverse cholesterol transport, ABCA3 is involved in surfactant formation. Objective: study the expression of ABCA1, ABCG1 and ABCA3 genes in smoking and COPD. Methods: the analysis was carried out on previously studied data sets (gene sets) obtained from the expression of Omnibus (GEO) genes. Results: gene expression in alveolar macrophages in smokers (set GSE2125) showed a significant decrease of gene expression of ABCA1, ABCG1, apolipoprotein L1, L3, L4. Gene expression in airway epithelium in smokers (sets GSE4498, GSE3320, GSE11906), showed no significant changes of gene expression ABCA1, ABCA3, ABCG1, apolipoprotein L3 and L6. Gene expression in alveolar epithelial type II cells in patients with COPD showed a significant decrease in the expression of genes ABCA1, ABCA3, ABCG1, apolipoprotein L1, L3, L6, increased expression of TLR3, TLR4 compared with healthy individuals (set GSE29133). Conclusion: smoking disrupts the reverse transport of cholesterol in macrophages and alveolar epithelium, which probably serves as a mechanism of inflammation progression, reduces the expression of apolipoprotein L, whose function is not well known, but is considered to be involved in immune processes.

A 3- to 8-fold stimulation of interleukin (IL)-8 gene expression by all-trans-retinoic acid (ATRA) was demonstrated in primary cultures of human and monkey tracheobronchial epithelial cells and BEAS-2B serum-sensitive cell line. The effect of ATRA on IL-8 gene expression is dose- and time-dependent. Using cycloheximide, it was observed that new protein synthesis was required for the stimulation. ATRA had no effect on IL-8 messenger RNA stability. A difference in nuclear run-on activity suggests that a transcriptional mechanism is involved in ATRA-enhanced IL-8 gene expression. Promoter-reporter gene transfection studies demonstrated ATRA enhanced IL-8 promoter activity, especially when cells were cotransfected with retinoic acid nuclear receptor-alpha expression vector. Deletion and site-directed mutagenesis analysis revealed the involvement of nuclear factor (NF)-kappaB binding site of the IL-8 gene in ATRA-enhanced promoter activity. Electrophoretic mobility shift assay (EMSA) demonstrated that ATRA enhanced DNA-NF-kappaB complex formation, especially with the p65 subunit. Western blot analysis demonstrated that ATRA did not enhance the protein amount of both the p50 and the p65 subunits in the nuclei. Because ATRA also enhances thioredoxin (TRX) gene expression, the effect of TRX on IL-8 gene expression was examined. IL-8 promoter activity was enhanced in transfected cells by the addition of TRX protein. Treatment of nuclear extracts with TRX also enhanced DNA- NF-kappaB complex formation as observed by EMSA, particularly the p65 subunit. Taking these data together, a novel mechanism is proposed in which ATRA activates promoter activity of IL-8 gene through TRX-dependent NF-kappaB activation.

Cigarette smoke deregulates several biological pathways by modulating gene expression in airway epithelial cells and altering the physiology of the airway epithelium. The effects of repeated exposures of electronic cigarette delivery systems (ENDS) on gene expression in airway epithelium are relatively unknown. In order to assess the effect of repeated exposures of ENDS, primary normal human bronchial epithelial (NHBE) cells grown at air-liquid interface (ALI) were exposed to cigarette and ENDS preparations daily for 10 days. Cigarette smoke preparations significantly altered gene expression in a dose-dependent manner compared to vehicle control, including genes linked to oxidative stress, xenobiotic metabolism, cancer pathways, epithelial-mesenchymal transition, fatty acid metabolism, degradation of collagen and extracellular matrix, O-glycosylation, and chemokines/cytokines, which are known pathways found to be altered in smokers. Conversely, ENDS preparations had minimal effect on transcriptional pathways. This study revealed that a sub-chronic exposure of primary NHBE cultures to cigarette and ENDS preparations differentially regulated genes and canonical pathways, with minimal effect observed with ENDS preparations compared to cigarette preparations. This study also demonstrates the versatility of primary NHBE cultures at ALI to evaluate repeat-dose exposures of tobacco products.

Transcriptome-wide association study of sex effects identifies sex-specific nasal epithelial gene expression profiles for total IgE.

The purpose of this study is to characterize glutathione S-transferase (GST) gene expression in airway epithelium both in vivo and in vitro. Immunohistochemical staining of nonhuman primate lungs of well-controlled healthy animals reveals the presence of alpha- and pi-class GST isoenzymes in ciliated bronchial epithelium. The stain of mu-GST antibody is either very low or absent in some of these monkey lungs. We observed that primary tracheobronchial epithelial (TBE) cells isolated from human and monkey pulmonary tissues maintain a relatively high level of GST enzymatic activity in culture, compared with various immortalized human TBE cell lines and other nonpulmonary cell lines. Northern blot analysis demonstrated the presence of mu-, pi-, and microsomal-GST messages but not the alpha-class message in cultures of primary TBE cells as well as in various human TBE cell lines. The expression of mu- and pi-class GST genes can be further regulated in culture by various environmental factors; however, most of these regulating factors are associated with TBE cell differentiation in culture. For instance, vitamin A treatment, which was shown to enhance mucous cell differentiation in vitro, stimulated the message levels of mu- and pi-class GST. Furthermore, plating cells on collagen gel substrata, which also enhanced mucous cell differentiation in culture, instead of plastic culture surface, enhanced total GST enzymatic activity by eightfold, and this enhancement is related to an increase in the expression of the pi-class GST gene. These results demonstrated that GST genes are differentially expressed and regulated by various environmental factors in primary TBE cells and various cell lines, and the regulation is correlated to the mucous cell differentiation in culture.

Mucus hypersecretion is an important characteristic feature of the pathogenesis of allergy. Although interleukin (IL)‐4 is known to be an inflammatory mediator in respiratory diseases, the mechanism by which IL‐4 induces MUC5AC gene expression has not been fully explored. The aim of this study was to investigate the mechanism by which IL‐4 induces MUC5AC gene expression in the airway. We examined the role of mitogen‐activated protein kinase (MAPK) signaling on MUC5AC gene expression in airway epithelium. We showed that phosphorylation of ERK1/2 increased after treatment of cells with IL‐4, whereas phosphorylation of p38 and JNK was not detected. In addition, pharmacologic and genetic inhibition of ERK1/2 abolished IL‐4‐induced MUC5AC gene expression. Moreover, we investigated the activation of p90 ribosomal S6 kinase 1 (RSK1) as a downstream signaling target of ERK1/2 in IL‐4 signaling. The activation of RSK1 was prevented by pretreatment with PD98059 or plasmid expressing a MEK1 dominant‐negative mutant. We also found that RSK1 mediated the IL‐4‐induced phosphorylation of cAMP response element‐binding protein (CREB) and the transcription of MUC5AC. Furthermore, the cAMP‐response element (CRE) in the MUC5AC promoter appears to be important for IL‐4‐induced MUC5AC gene expression in NCI‐H292 cells. J. Cell. Biochem. 108: 974–981, 2009. © 2009 Wiley‐Liss, Inc.

Persistent infection with Pseudomonas aeruginosa increases interleukin-8 (IL-8) levels and causes dense neutrophil infiltrations in the airways of patients with chronic airway diseases. Recently, we have reported that nitrite reductase from P. aeruginosa induces the production of IL-8 in respiratory cells, including bronchial epithelial cells. To determine the molecular mechanism(s) of nitrite reductase-induced IL-8 expression in respiratory cells, A549 epithelial cells were transfected with plasmids containing serial deletions of the 5'-flanking region of the IL-8 gene and then exposed to nitrite reductase. Nitrite reductase significantly enhanced IL-8 gene promoter-driven reporter activity. This increased IL-8 gene expression was inhibited by mutating the nuclear factor-kappaB (NF-kappaB) binding element. Nitrite reductase enhanced nuclear localization of the NF-kappaB binding complex. Furthermore, nitrite reductase induced the degradation of IkappaBalpha, the major cytoplasmic inhibitor of NF-kappaB, and the expression of IkappaBalpha mRNA. These data support the critical role of the activation of NF-kappaB in nitrite reductase-induced IL-8 gene expression in airway epithelium.

Persistent infection with Pseudomonas aeruginosa increases interleukin-8 (IL-8) levels and causes dense neutrophil infiltrations in the airways of patients with chronic airway diseases. Recently, we have reported that nitrite reductase from P. aeruginosa induces the production of IL-8 in respiratory cells, including bronchial epithelial cells. To determine the molecular mechanism(s) of nitrite reductase-induced IL-8 expression in respiratory cells, A549 epithelial cells were transfected with plasmids containing serial deletions of the 5'-flanking region of the IL-8 gene and then exposed to nitrite reductase. Nitrite reductase significantly enhanced IL-8 gene promoter-driven reporter activity. This increased IL-8 gene expression was inhibited by mutating the nuclear factor-kappaB (NF-kappaB) binding element. Nitrite reductase enhanced nuclear localization of the NF-kappaB binding complex. Furthermore, nitrite reductase induced the degradation of IkappaBalpha, the major cytoplasmic inhibitor of NF-kappaB, and the expression of IkappaBalpha mRNA. These data support the critical role of the activation of NF-kappaB in nitrite reductase-induced IL-8 gene expression in airway epithelium.

<b>Background:</b> Expression quantitative trait loci (eQTL) analyses help to understand the function of disease-associated genetic risk factors. However, studies based on bulk RNA-seq data are constrained by the lack of cell-type resolution. <b>Aim:</b> We aim to identify cell-type-specific regulation of asthma-associated genetic variants on gene expression in airway epithelium by eQTL deconvolution. <b>Methods:</b> Matched genotype data and bulk RNAseq data of bronchial biopsy were available for 146 subjects. We firstly estimated the proportions of cell subpopulations of bronchial biopsy using marker genes identified in a single cell (sc-) RNAseq dataset of bronchial biopsy (N=11). Then we used Decon-eQTL (Aguirre-Gamboa R, et al. BMC bioinformatics, 2020, 21(1): 1-23) to identify cell type cis-eQTLs of asthma-associated variants for three main epithelial cell types (ciliated cells, basal cells, and secretory cells). We replicated these results using independent single-cell eQTL data from bronchial biopsies (N=28). <b>Results:</b> We identified 14 cell-type cis-eQTLs (P-value &lt; 0.001) and four were replicated by single-cell eQTL method with P-value &lt;0.05, including rs4749894-<i>CALML5</i> in basal cells, and rs8103278-<i>FOXA3</i> in secretory cells. <i>CALML5</i> was associated with the differentiation of keratinocytes, and <i>FOXA3</i> played an important role in goblet cell metaplasia that is related to asthma development. <b>Conclusions:</b> We identified cell type eQTL by an eQTL deconvolution method using bulk RNA-seq data, and the results can be replicated by single-cell eQTL method. eQTL deconvolution analyses may be a powerful tool to better understanding cell-type-specific molecular mechanisms of asthma development.