Exposure of bronchial airway epithelial cells to elexacaftor/tezacaftor/ivacaftor, Pseudomonas aeruginosa strain PA14 and outer membrane vesicles
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ABSTRACT:
ORGANISM(S): Homo sapiens
PROVIDER: GSE268718 | GEO | 2024/06/25
REPOSITORIES: GEO
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Project description:Cystic fibrosis (CF) is a life-shortening disease caused by mutations in the cystic fibrosis transmembrane conductance regulator (CFTR) gene. Although bacterial lung infection and the resulting inflammation cause most of the morbidity and mortality, how loss of CFTR first disrupts airway host defense has remained uncertain. We asked what abnormality impairs elimination when a bacterium lands on the pristine surface of a newborn CF airway? To investigate this defect, we interrogated the viability of individual bacteria immobilized on solid grids and placed on the airway surface. As a model we studied CF pigs, which spontaneously develop hallmark features of CF lung disease. At birth, their lungs lack infection and inflammation, but have a reduced ability to eradicate bacteria. Here we show that in newborn wild-type pigs, the thin layer of airway surface liquid (ASL) rapidly killed bacteria in vivo, when removed from the lung, and in primary epithelial cultures. Lack of CFTR reduced bacterial killing. We found that ASL pH was more acidic in CF, and reducing pH inhibited the antimicrobial activity of ASL. Reducing ASL pH diminished bacterial killing in wild-type pigs, and increasing ASL pH rescued killing in CF pigs. These results directly link the initial host defense defect to loss of CFTR, an anion channel that facilitates HCO3- transport. Without CFTR, airway epithelial HCO3- secretion is defective, ASL pH falls and inhibits antimicrobial function, and thereby impairs killing of bacteria that enter the newborn lung. These findings suggest that increasing ASL pH might prevent the initial infection in patients with CF and that assaying ASL pH or bacterial killing could report on the benefit of therapeutic interventions. 11 samples of trachea primary airway epithelial cultures representing CFTR+/+ and CFTR-/- pigs. Pig samples representing 14 bronchus and 12 trachea tissue samples submitted in GSE21071.
2012-03-29 | E-GEOD-36906 | biostudies-arrayexpress
Project description:Cystic fibrosis (CF) is an inherited, multi-system disease caused by dysfunction of the cystic fibrosis transmembrane conductance regulator (CFTR) protein, a ubiquitous ion channel important for epithelial hydration. A direct consequence of this dysfunction is impaired mucociliary clearance, chronic airway infection and a persistent neutrophilic inflammatory response that results in progressive loss of lung function, development of respiratory failure and premature death. Partial restoration of CFTR function is now possible for most CF patients through mutation specific CFTR modulators. Ivacaftor monotherapy produces significant clinical improvement in CF patients with the G511D mutation. Dual therapy, combining ivacaftor with lumacaftor or tezacaftor, results in modest clinical improvements in patients homozygous for F508del. More recently, triple therapy with elexacaftor/tezacaftor/ivacaftor (ETI) has led to dramatic improvements in lung function and quality of life in patients homozygous and heterozygous for F508del. Sputum proteomics is a powerful research technique capable of identifying important airway disease mechanisms by interrogating the proteome, an entire set of proteins within biological samples. It has confirmed the central role of neutrophilic immune dysregulation in CF and non-CF bronchiectasis, particularly involving the release of antimicrobial proteins and neutrophil-extracellular traps (NETs), and through impaired anti-inflammatory mechanisms. These processes produce distinct molecular signatures within the sputum proteome that become increasingly abnormal with chronic airway infection and progressive lung disease severity. In CF patients, airway and systemic inflammatory cytokines potentially related to these signatures reduce with the various forms of CFTR modulation. To date, no studies of ETI therapy in CF lung disease have assessed large-scale change in protein expression using untargeted proteomics. We hypothesised that ETI therapy would shift the sputum proteome toward health, potentially normalising airway biology in people with CF. The objectives of this study were to investigate changes in the CF sputum proteome with the introduction of ETI, correlate these with changes in clinical markers of disease severity, and make comparisons with the sputum proteome in healthy controls and in repeat samples from CF patients not suitable for ETI therapy. We also explored which molecular pathways associated with CF lung disease did not change with ETI.
2023-11-14 | PXD041950 | Pride
Project description:We studied miRNAs and their gene targets affecting SARS-CoV-2 pathogenesis in CF airway epithelial cell models in response to TGF-β1. Small RNAseq in CF human bronchial epithelial cell line treated with TGF-β1 and miRNA profiling characterized TGF-β1 effects on the SARS-CoV-2 pathogenesis pathways. Among the effectors, we identified and validated two miRNAs targeting ACE2 mRNA using different CF and non-CF human bronchial epithelial cell models. We have shown that TGF-β1 inhibits ACE2 expression by miR-136-3p and miR-369-5p. ACE2 levels were higher in cells expressing F508del-CFTR, compared to wild-type(WT)-CFTR and TGF-β1 inhibited ACE2 in both cell types. The ACE2 protein levels were still higher in CF, compared to non-CF cells after TGF-β1 treatment. TGF-β1 prevented the functional rescue of F508del-CFTR by ETI in primary human bronchial epithelial cells while ETI did not prevent the TGF-β1 inhibition of ACE2 protein. Finally, TGF-β1 reduced binding of ACE2 to the recombinant monomeric spike RBD. Our results may help to explain, at least in part, the role of TGF-β1 on the SARS-CoV-2 entry via ACE2 in the CF and non-CF airway.
2023-04-01 | GSE226758 | GEO
Project description:Cystic fibrosis (CF) is a life-shortening disease caused by mutations in the cystic fibrosis transmembrane conductance regulator (CFTR) gene. Although bacterial lung infection and the resulting inflammation cause most of the morbidity and mortality, how loss of CFTR first disrupts airway host defense has remained uncertain. We asked what abnormality impairs elimination when a bacterium lands on the pristine surface of a newborn CF airway? To investigate this defect, we interrogated the viability of individual bacteria immobilized on solid grids and placed on the airway surface. As a model we studied CF pigs, which spontaneously develop hallmark features of CF lung disease. At birth, their lungs lack infection and inflammation, but have a reduced ability to eradicate bacteria. Here we show that in newborn wild-type pigs, the thin layer of airway surface liquid (ASL) rapidly killed bacteria in vivo, when removed from the lung, and in primary epithelial cultures. Lack of CFTR reduced bacterial killing. We found that ASL pH was more acidic in CF, and reducing pH inhibited the antimicrobial activity of ASL. Reducing ASL pH diminished bacterial killing in wild-type pigs, and increasing ASL pH rescued killing in CF pigs. These results directly link the initial host defense defect to loss of CFTR, an anion channel that facilitates HCO3- transport. Without CFTR, airway epithelial HCO3- secretion is defective, ASL pH falls and inhibits antimicrobial function, and thereby impairs killing of bacteria that enter the newborn lung. These findings suggest that increasing ASL pH might prevent the initial infection in patients with CF and that assaying ASL pH or bacterial killing could report on the benefit of therapeutic interventions.
2012-03-30 | GSE36906 | GEO
Project description:Cystic Fibrosis Related Diabetes (CFRD), the main co-morbidity in Cystic Fibrosis (CF), is associated with higher rates of lung function decline. We hypothesize that airway epithelial barrier function is impaired in CF and is further exacerbated under hyperglycemia, worsening pulmonary outcomes. Using 16HBE cells as a model cell line, we studied the effects of hyperglycemia in airway epithelial barrier function. Results show increased paracellular dye flux in CF cells in response to insulin treatment under hyperglycemia, suggesting impaired barrier integrity. Gene expression experiments identified Claudin-4 (CLDN4) as a key tight junction protein dysregulated in CF cells. Further investigation into CLDN4 protein localization by confocal microscopy showed that CLDN4 was tightly localized at tight junctions in WT cells and localization did not change under hyperglycemia. ln contrast, CLDN4 was less well-localized in CF cells at normal glucose and localization was worsened in CF cells conditioned to hyperglycemia. Treatment with highly effective modulator compounds (ETI) reversed this trend, and CFTR rescue by ETI in CF cells was not affected by insulin treatment or hyperglycemia. Bulk RNA sequencing showed differences in transcriptional responses in CF compared to WT cells under normal or high glucose, highlighting PTPRG as a promising target for further investigation.
2024-06-03 | GSE268909 | GEO
Project description:Speciation leads to adaptive changes in organ cellular anatomy and physiology. These evolutionary changes create challenges for studying rare cell type functions that diverge between human and mice. Rare CFTR-rich pulmonary ionocytes exist throughout the cartilaginous airways of humans, but limited presence and divergent biology in the proximal trachea of mice has prevented the use of traditional transgenic models to elucidate ionocyte functions in the airway. Here we describe the creation and use of novel conditional genetic ferret models to dissect pulmonary ionocyte biology and function by enabling ionocyte lineage tracing (FOXI1-CreERT2::ROSA-TG), ionocyte ablation (FOXI1-KO), and ionocyte-specific deletion of CFTR (FOXI1-CreERT2::CFTRL/L). By comparing these models to cystic fibrosis (CF) ferrets, we demonstrate that ionocytes control airway surface liquid (ASL) absorption, secretion, pH, and mucus viscosity—leading to reduced ASL volume and impaired mucociliary clearance in CF, FOXI1-KO, and FOXI1-CreERT2::CFTRL/L ferrets. These processes were regulated by CFTR- dependent ionocyte transport of Cl– and HCO3–, as determined electrophysiologically and by single-cell imaging with a conditionally activated halide fluorescent sensor. Single-cell transcriptomics, using pulse-seq of lineage-traced ionocyte-enriched cultures, revealed three subtypes of pulmonary ionocytes with unique biologic functions and a common rare cell progenitor of ionocytes, tuft, and neuroendocrine cells. Thus, rare pulmonary ionocytes perform critical CFTR-dependent functions in the proximal airway that are hallmark features of CF airway disease. These studies provide a road map for using conditional genetics in the first non- rodent mammal to address gene function, cell biology, and disease processes that have greater evolutionary conservation between humans and ferrets.
2023-08-01 | GSE233654 | GEO
Project description:Our laboratory has held a long interest in the glycosylation changes seen on the surface of airway epithelia of patients with the disease cystic fibrosis (CF). Experiments from our laboratory have detailed a CF glycosylation phenotype of increased Fuca1,3/4 and decreased Fuca1,2 and sialic acid on the surfaces of immortalized and primary CF cells compared to non-CF cells. Further, we have shown that gene transfer and subsequent expression of a wild type CF plasmid in CF airway cells results in correction or reversal of this glycosylation phenotype. We hypothesize that the changes in glycosylation seen in CF cells are key in the pathophysiology of the cystic fibrosis airway disease. For example, it has been shown that Pseudomonas aeruginosa, a bacterium that has a predilection for colonizing CF airways, adheres to asialylated glycolipids and glycoconjugates with terminal Fuca1,3/4. One focus of our laboratory is to elucidate the etiology of the glycosylation changes seen in CF cells and the mechanism by which these changes are reversed by wild type CFTR gene transfer. We propose to study the gene expression of immortalized and primary CF and non-CF airway epithelial cells: 1. CF/T43 vs. BEAS-2B cells. These are two widely used immortalized airway cell lines that we have used extensively in the past. 2. C38 cells; C38 cells are IB3 cells expressing wtCFTR. The experimental focus is to elucidate the etiology of the glycosylation changes seen in Cystic Fibrosis (CF) cells and the mechanism by which these changes are reversed by wild type CFTR gene transfer. To do so, the gene expression of immortalized and primary CF and non-CF airway epithelial cells were compared and studied. Cell lines used were CF/T43 and BEAS-2B, both widely used immortalized airway cell lines. Other cell lines studied included C38 cell lines (clonal derivatives of IB3 cells expressing wtCFTR).
2011-01-07 | E-GEOD-26482 | biostudies-arrayexpress
Project description:Purpose: To identify the cell types that contribute to CFTR expression and function within the proximal-distal axis of the normal human lung. Methods: Single cell RNA-seq (scRNA-seq) was performed on freshly isolated human large and small airway epithelial cells. Single cell-based RNA in situ hybridization (scRNA-ISH) and quantitative RT-PCR (scqRT-PCR) were performed for validation. In vitro culture systems correlated CFTR function to cell types. Lentiviruses were used for cell-type specific transduction of wild-type CFTR in CF cells. Results: scRNA-seq identified secretory cells as dominating CFTR expression in normal human large and particularly small airways, followed by basal cells. Ionocytes expressed the highest CFTR levels but were rare, while expression in ciliated cells was infrequent and low. scRNA-ISH, and scqRT-PCR confirmed the scRNA-seq findings. CF lungs exhibited distributions of CFTR and ionocytes similar to normal controls. CFTR-mediated Cl- secretion tracked secretory cell, not ionocyte, densities. Further, the nucleotide-purinergic regulatory system that controls CFTR-mediated hydration was associated with secretory cells. Lentiviral transduction of wild-type CFTR produced CFTR-mediated Cl- secretion in CF airway secretory cells but not ciliated cells. Conclusions: Secretory cells dominate CFTR expression and function in human airway superficial epithelia. CFTR therapies may need to restore CFTR function to multiple cell types, with a focus on secretory cells.
2020-12-16 | GSE160673 | GEO
Project description:Purpose: To identify the cell types that contribute to CFTR expression and function within the proximal-distal axis of the normal human lung. Methods: Single cell RNA-seq (scRNA-seq) was performed on freshly isolated human large and small airway epithelial cells. Single cell-based RNA in situ hybridization (scRNA-ISH) and quantitative RT-PCR (scqRT-PCR) were performed for validation. In vitro culture systems correlated CFTR function to cell types. Lentiviruses were used for cell-type specific transduction of wild-type CFTR in CF cells. Results: scRNA-seq identified secretory cells as dominating CFTR expression in normal human large and particularly small airways, followed by basal cells. Ionocytes expressed the highest CFTR levels but were rare, while expression in ciliated cells was infrequent and low. scRNA-ISH, and scqRT-PCR confirmed the scRNA-seq findings. CF lungs exhibited distributions of CFTR and ionocytes similar to normal controls. CFTR-mediated Cl- secretion tracked secretory cell, not ionocyte, densities. Further, the nucleotide-purinergic regulatory system that controls CFTR-mediated hydration was associated with secretory cells. Lentiviral transduction of wild-type CFTR produced CFTR-mediated Cl- secretion in CF airway secretory cells but not ciliated cells. Conclusions: Secretory cells dominate CFTR expression and function in human airway superficial epithelia. CFTR therapies may need to restore CFTR function to multiple cell types, with a focus on secretory cells.
2020-12-16 | GSE160674 | GEO
Project description:Background: CFTR modulators will decrease some etiologies of inflammation in the CF airway; however, current data indicate that non-resolving airway infection and inflammation will persist in individuals with CF and chronic bacterial infections. Thus, identification of therapies that diminish CF airway inflammation without allowing unrestrained bacterial growth remains a critical research goal. Novel strategies for combatting deleterious airway inflammation require a better understanding of the cellular contributions to chronic CF airway disease, and how cells change after initiation of CFTR modulator therapy. Peripheral blood monocytes, which traffic to the CF airway, can develop both pro-inflammatory and inflammation-resolving phenotypes, and represent intriguing targets for focused therapies to dampen CF airway inflammation. Material and Methods: In order to characterize the inflammatory phenotype of CF blood monocytes, and how these cells change after initiation of CFTR modulator therapy, we studied adults (n=10) with CF, chronic airway infections, and the CFTR-R117H mutations before and 7 days after initiation of ivacaftor. Transcriptomes of freshly isolated blood monocytes were interrogated by RNA-sequencing (RNA-seq). Plasma concentrations of cytokines and chemokines were evaluated by multiplex ELISA. Results: RNAseq identified approximately 50 monocyte genes for which basal expression was significantly changed in all 10 subjects after 7 days of ivacaftor. Of these, the majority were increased in expression post ivacaftor, including many genes traditionally associated with enhanced inflammation and immune responses. Pathway analyses confirmed that transcriptional pathways were overwhelmingly up-regulated in monocytes after 7 days of ivacaftor, including transcriptional modules associated with immunity, cell cycle, oxidative phosphorylation, and the unfolded protein response. Ivacaftor increased plasma concentrations of CXCL2, a neutrophil chemokine secreted by monocytes and macrophages, and CCL2, a monocyte chemokine. These results demonstrate that ivacaftor causes acute changes in blood monocytes and plasma chemokines, and suggest that increased monocyte inflammatory signals and potentially improved trafficking to the lung contribute to changes in airway inflammation in people taking CFTR modulators. To our knowledge, this is the first report investigating the transcriptomic response of isolated blood monocytes in CF subjects.
2020-07-31 | GSE148076 | GEO