A revised airway epithelial hierarchy includes CFTR-expressing ionocytes
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ABSTRACT:
ORGANISM(S): Mus musculus
PROVIDER: GSE103354 | GEO | 2018/07/30
REPOSITORIES: GEO
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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:The functions of epithelial tissues are dictated by the types, abundance, and distribution of the differentiated cells they contain. Attempts to restore tissue function after damage require knowledge of how physiological tasks are distributed among cell types, and how cell states vary between homeostasis, injury/repair, and disease. In the conducting airway, a heterogeneous basal cell population gives rise to specialized luminal cells that perform mucociliary clearance. We performed single cell profiling of human bronchial epithelial cells and mouse tracheal epithelial cells to obtain a comprehensive picture of cell types in the conducting airway and their behavior in homeostasis and regeneration. Our analysis reveals cell states that represent known and novel cell populations, delineates their heterogeneity, and identifies distinct differentiation trajectories during homeostasis and tissue repair. Finally, we identified a novel, rare cell type, which we call the "pulmonary ionocyte", that co-expresses FOXI1, multiple subunits of the V-ATPase, and CFTR, the gene mutated in cystic fibrosis (CF). Using immunofluorescence, signaling pathway modulation, and electrophysiology, we show that Notch signaling is necessary and FOXI1 expression sufficient to drive the production of the pulmonary ionocyte, and that the pulmonary ionocyte is a major source of CFTR activity in the conducting airway epithelium.
2018-08-01 | GSE102580 | GEO
Project description:Salivary glands are essential structures that secrete saliva to the oral cavity and maintain oral health. Development of salivary glands in mice and humans is controlled by mesenchymally expressed fibroblast growth factor-10 (FGF10). Using single cell RNA-seq atlas of the salivary gland and a tamoxifen inducible Fgf10CreERT2:R26-tdTomato mouse we show that FGF10pos cells are exclusively mesenchymal until postnatal day 5 (P5), but after P7, there is a switch in expression and only epithelial FGF10pos cells are observed after P15. Further RNAseq analysis of sorted mesenchymal and epithelial FGF10pos cells shows that the epithelial FGF10pos populations express the hallmark of ancient ionocyte signature Foxi1, Foxi2, Ascl3 and the cystic fibrosis transmembrane conductance regulator (Cftr). We propose that epithelial FGF10pos cells are specialized salivary gland ionocytes that are important for the ionic modification of saliva. In addition, they maintain FGF10-dependent glands homeostasis via communication with FGFR2b-expressing epithelial progenitor and myoepithelial cells
2022-04-12 | GSE188904 | GEO
Project description:To interrogate the intrinsic effect of CFTR on airway epithelium; to interrogate the presence of FOXi1+ASLC3+ ionocyte population. Description of samples:1567 (F508del CF patient of rapid progressive phenotye) and 1566 (isogenic CFTR-repaired pair). iPSC-derived airway epithelium, s/p D15CD47hi/CD26lo sort, 210DCYI media x
2023-03-02 | GSE203007 | GEO
Project description:We have compared gene expression in human nasal brushing cells from 19 cystic fibrosis (CF) patients and 19 healthy controls using a 5.2K cDNA microarray. Our aim is to identify new disease biomarkers for the Cystic Fibrosis Gene Therapy Consortium. These markers will be used to report more effectively on the response to the administration of gene therapy in vivo. Cystic Fibrosis is a recessive genetic disease caused by mutations in the cystic fibrosis conductance regulator (CFTR) gene which encodes a chloride ion channel. The most common mutation is the ∆F508 mutation, present on 70% of CF chromosomes in Caucasian populations. The disease affects many organs in the body such as the pancreas, liver, sweat glands, small intestine and reproductive tracts but is most commonly associated with progressive, inflammatory lung disease. The current average life expectancy of CF patients is 35 years. Gene therapy is being developed as a treatment for CF airway disease, however, means of measuring the efficiency and efficacy of gene therapy in vivo are lacking. This is mainly due to the difficulty in measuring the chloride conductance of CFTR in cells and tissues. Furthermore, clinical assays for measuring improvements in lung function are insensitive. Surrogate markers of inflammation and CFTR function will therefore be important for the effective assessment of gene therapy in vivo. We have analysed gene expression in human nasal epithelium as this is considered an accessible surrogate for the conducting airways where disease manifests in the majority of patients. Additionally, this tissue will be sampled in clinical trials.
2011-04-30 | E-MEXP-436 | 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:Cystic fibrosis, the most commonly inherited lethal pulmonary disorder in Caucasians, is caused by mutations in the cystic fibrosis transmembrane conductance regulator gene (CFTR). To identify genomic responses to the presence or absence of CFTR in pulmonary tissues in vivo, microarray analyses of lung mRNAs were performed on whole lung tissue from mice lacking (CFTR(-)) or expressing mouse CFTR (CFTR(+)). Whereas the histology of lungs from CFTR(-) and CFTR(+) mice was indistinguishable, statistically significant increases in the relative abundance of 29 and decreases in 25 RNAs were identified by RNA microarray analysis. Of RNAs whose expression was consistently altered by the absence of CFTR, functional classes of genes influencing gene transcription, inflammation, intracellular trafficking, signal transduction, and ion transport were identified. RNAs encoding the transcription factor CCAAT enhancer-binding protein (CEBP) delta and interleukin (IL) 1beta, both known to regulate CFTR expression, were induced, perhaps indicating adaptation to the lack of CFTR. RNAs mediating lung inflammation including calgranulin-S100 family members, IL-1beta and IL-4, were increased. Likewise, expression of several membrane transport proteins that interact directly with CFTR were increased, suggesting that CFTR-protein complexes initiate genomic responses. Absence of CFTR influenced the expression of genes modulating diverse pulmonary cell functions that may ameliorate or contribute to the pathogenesis of CF. Lungs from sex-matched littermates at 3, 6, and 11 weeks of agewere carefully dissected and the conducting airways and mediastinal structures removed.
2007-11-27 | E-GEOD-4513 | biostudies-arrayexpress
Project description:Production of functional proteins requires multiple steps including gene transcription and post-translational processing. MicroRNAs (miRNA) can regulate individual stages of these processes. Despite the importance of the cystic fibrosis transmembrane conductance regulator (CFTR) channel for epithelial anion transport, how its expression is regulated remains uncertain. We discovered that microRNA-138 regulates CFTR expression through its interactions with the transcriptional regulatory protein SIN3A. Treating airway epithelia with a miR-138 mimic increased CFTR mRNA and also enhanced CFTR abundance and transepithelial Cl- permeability independently of elevated mRNA levels. A miR-138 anti-miR had the opposite effects. Importantly, miR-138 altered the expression of many genes encoding proteins that associate with CFTR and may influence its biosynthesis. The most common CFTR mutation, M-NM-^TF508, causes protein misfolding, degradation, and cystic fibrosis. Remarkably, manipulating the miR-138 regulatory network also improved biosynthesis of CFTR-M-NM-^TF508 and restored Cl- transport to cystic fibrosis airway epithelia. This novel miRNA-regulated network directs gene expression from the chromosome to the cell membrane, indicating that an individual miRNA can control a cellular process broader than previously recognized. This discovery also provides new therapeutic avenues for restoring CFTR function to cells affected by the most common cystic fibrosis mutation. 12 samples of Calu-3 cells representing different interventions.
2012-09-04 | E-GEOD-38956 | biostudies-arrayexpress
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