Project description:About 50% of patients with asthma exhibit chronic airway inflammation driven by the type 2 cytokines interleukin (IL)-4, IL-5, and IL-13. These patients with type 2-high asthma experience more allergic symptoms, greater airway hyperresponsiveness, and more severe mucus obstruction than patients with type 2-low asthma. Mouse models of asthma have shown that much of the airway dysfunction in these models can be generated by IL-13 stimulation of the airway epithelium alone. Both in vivo mouse model studies and in vitro studies of human mucociliary airway epithelial cultures have shown that IL-13 induces cellular remodeling of the airway epithelium through proliferation-independent transdifferentiation processes. In both humans and mice, IL-13 stimulation of the airway epithelium results in generation of hypersecretory mucin 5AC (MUC5AC)-expressing mucus cells. Whereas club cells have been shown to be the source of these mucin 5AC-positive mucus cells in mice, the origin of these mucus cells in humans is unclear. In humans, chronic IL-13 stimulation appears to result in loss of ciliated cells. Moreover, IL-13 stimulation can block ciliated cell differentiation from human basal airway epithelial cells. Coincident with IL-13 cellular remodeling are reported decreases in mucociliary transport and ciliary beat frequency. These IL-13-mediated changes in mucociliary function are accompanied by disorganization of cilia, a decrease in the ratio of mucin 5B (MUC5B) to mucin 5AC, and mucus gel tethering to the epithelial surface by mucin 5AC. These airway epithelial responses to IL-13 are mediated by multiple transcription factors, including signal transducer and activator of transcription-6 (STAT6), SAM pointed domain-containing Ets transcription factor (SPDEF), Forkhead box A2 (FOXA2), and Forkhead box J1 (FOXJ1). In addition, analysis of RNA-sequencing data derived from airway epithelial cells shows how IL-13 stimulation promotes broad changes in gene expression across the transcriptome. These results reveal the plastic nature of airway epithelial cells that enables the epithelium to undergo remodeling and functional shifts in response to IL-13 stimulation. With use of new technology, future studies should lead to greater understanding of how IL-13 and other stimuli of disease bring about airway epithelial remodeling, which may aid in the development of therapies that ameliorate airway dysfunction in asthma and other diseases.
Project description:Most of the airways of the human lung are lined by an epithelium made up of ciliated and secretory luminal cells and undifferentiated basal progenitor cells. The integrity of this epithelium and its ability to act as a selective barrier are critical for normal lung function. In other epithelia, there is evidence that transcription factors of the evolutionarily conserved grainyheadlike (GRHL) family play key roles in coordinating multiple cellular processes required for epithelial morphogenesis, differentiation, remodeling, and repair. However, only a few target genes have been identified, and little is known about GRHL function in the adult lung. Here we focus on the role of GRHL2 in primary human bronchial epithelial cells, both as undifferentiated progenitors and as they differentiate in air-liquid interface culture into an organized mucociliary epithelium with transepithelial resistance. Using a dominant-negative protein or shRNA to inhibit GRHL2, we follow changes in epithelial phenotype and gene transcription using RNA sequencing or microarray analysis. We identify several hundreds of genes that are directly or indirectly regulated by GRHL2 in both undifferentiated cells and air-liquid interface cultures. Using ChIP sequencing to map sites of GRHL2 binding in the basal cells, we identify 7,687 potential primary targets and confirm that GRHL2 binding is strongly enriched near GRHL2-regulated genes. Taken together, the results support the hypothesis that GRHL2 plays a key role in regulating many physiological functions of human airway epithelium, including those involving cell morphogenesis, adhesion, and motility.
Project description:Pseudostratified airway epithelium of the lung is composed of polarized ciliated and secretory cells maintained by basal stem/progenitor cells. An important question is how lineage choice and differentiation are coordinated with apical-basal polarity and epithelial morphogenesis. Our previous studies indicated a key integrative role for the transcription factor Grainyhead-like 2 (Grhl2). In this study, we present further evidence for this model using conditional gene deletion during the regeneration of airway epithelium and clonal organoid culture. We also use CRISPR/Cas9 genome editing in primary human basal cells differentiating into organoids and mucociliary epithelium in vitro. Loss of Grhl2 inhibits organoid morphogenesis and the differentiation of ciliated cells and reduces the expression of both notch and ciliogenesis genes (Mcidas, Rfx2, and Myb) with distinct Grhl2 regulatory sites. The genome editing of other putative target genes reveals roles for zinc finger transcription factor Znf750 and small membrane adhesion glycoprotein in promoting ciliogenesis and barrier function as part of a network of genes coordinately regulated by Grhl2.
Project description:Airway basal stem cells are the progenitor cells within the airway that exhibit the capacity to self-renew and give rise to multiple types of differentiated airway epithelial cells. This stem cell-derived epithelium displays organized architecture with functional attributes of the airway mucosa. A protocol has been developed to culture and expand human airway basal stem cells while preserving their stem cell properties and capacity for subsequent mucociliary differentiation. This achievement presents a previously unrealized opportunity to maintain a durable supply of progenitor cells derived from healthy donors to differentiate into human primary airway epithelium for cellular and molecular-based studies. Further, basal stem cells can be harvested from patients with a specific airway disease, such as cystic fibrosis, enabling investigation of potentially altered behavior of disease-specific cells in the appropriate context of the airway mucosa. Here we describe, in detail, a protocol for the serial expansion of airway basal stem cells to enable the generation of nearly unlimited airway basal cells that can be stored and readily available for subsequent culturing and differentiation. In addition, we describe culturing and differentiation of airway basal stem cells on permeable transwell filters at air-liquid interface to create functional mucociliary pseudostratified polarized airway epithelial mucosa.
Project description:<br /> Pseudostratified airway epithelium of the lung is composed of polarized ciliated and secretory cells maintained by basal stem/progenitor cells. An important question is how lineage choice and differentiation are coordinated with apical–basal polarity and epithelial morphogenesis. Our previous studies indicated a key integrative role for the transcription factor Grainyhead-like 2 (Grhl2). In this study, we present further evidence for this model using conditional gene deletion during the regeneration of airway epithelium and clonal organoid culture. We also use CRISPR/Cas9 genome editing in primary human basal cells differentiating into organoids and mucociliary epithelium in vitro. Loss of Grhl2 inhibits organoid morphogenesis and the differentiation of ciliated cells and reduces the expression of both notch and ciliogenesis genes (<br /> <jats:italic>Mcidas</jats:italic><br /> ,<br /> <jats:italic>Rfx2</jats:italic><br /> , and<br /> <jats:italic>Myb</jats:italic><br /> ) with distinct Grhl2 regulatory sites. The genome editing of other putative target genes reveals roles for zinc finger transcription factor Znf750 and small membrane adhesion glycoprotein in promoting ciliogenesis and barrier function as part of a network of genes coordinately regulated by Grhl2.<br />
Project description:Current methods to replace damaged upper airway epithelium with exogenous cells are limited. Existing strategies use grafts that lack mucociliary function, leading to infection and the retention of secretions and keratin debris. Strategies that regenerate airway epithelium with mucociliary function are clearly desirable and would enable new treatments for complex airway disease.Here, we investigated the influence of the extracellular matrix (ECM) on airway epithelial cell adherence, proliferation and mucociliary function in the context of bioengineered mucosal grafts. In vitro, primary human bronchial epithelial cells (HBECs) adhered most readily to collagen IV. Biological, biomimetic and synthetic scaffolds were compared in terms of their ECM protein content and airway epithelial cell adherence.Collagen IV and laminin were preserved on the surface of decellularised dermis and epithelial cell attachment to decellularised dermis was greater than to the biomimetic or synthetic alternatives tested. Blocking epithelial integrin ?2 led to decreased adherence to collagen IV and to decellularised dermis scaffolds. At air-liquid interface (ALI), bronchial epithelial cells cultured on decellularised dermis scaffolds formed a differentiated respiratory epithelium with mucociliary function. Using in vivo chick chorioallantoic membrane (CAM), rabbit airway and immunocompromised mouse models, we showed short-term preservation of the cell layer following transplantation.Our results demonstrate the feasibility of generating HBEC grafts on clinically applicable decellularised dermis scaffolds and identify matrix proteins and integrins important for this process. The long-term survivability of pre-differentiated epithelia and the relative merits of this approach against transplanting basal cells should be assessed further in pre-clinical airway transplantation models.
Project description:Mucociliary epithelia provide a first line of defense against pathogens. Impaired regeneration and remodeling of mucociliary epithelia are associated with dysregulated Wnt/?-catenin signaling in chronic airway diseases, but underlying mechanisms remain elusive, and studies yield seemingly contradicting results. Employing the Xenopus mucociliary epidermis, the mouse airway, and human airway Basal cells, we characterize the evolutionarily conserved roles of Wnt/?-catenin signaling in vertebrates. In multiciliated cells, Wnt is required for cilia formation during differentiation. In Basal cells, Wnt prevents specification of epithelial cell types by activating ?N-TP63, a master transcription factor, which is necessary and sufficient to mediate the Wnt-induced inhibition of specification and is required to retain Basal cells during development. Chronic Wnt activation leads to remodeling and Basal cell hyperplasia, which are reversible in vivo and in vitro, suggesting Wnt inhibition as a treatment option in chronic lung diseases. Our work provides important insights into mucociliary signaling, development, and disease.
Project description:Na+/H+ exchangers (NHEs) represent a highly conserved family of ion transporters that regulate pH homeostasis. NHEs as well as other proton transporters were previously linked to the regulation of the Wnt signaling pathway, cell polarity signaling, and mucociliary function. Furthermore, mutations in the gene SLC9A3 (encoding NHE3) were detected as additional risk factors for airway infections in cystic fibrosis patients. Here, we used the Xenopus embryonic mucociliary epidermis as well as human airway epithelial cells (HAECs) as models to investigate the functional roles of NHEs in mucociliary development and regeneration. In Xenopus embryos, NHEs 1-3 were expressed during epidermal development, and loss of NHE function impaired mucociliary clearance in tadpoles. Clearance defects were caused by reduced cilia formation, disrupted alignment of basal bodies in multiciliated cells (MCCs), and dysregulated mucociliary gene expression. These data also suggested that NHEs may contribute to the activation of Wnt signaling in mucociliary epithelia. In HAECs, pharmacological inhibition of NHE function also caused defective ciliation and regeneration in airway MCCs. Collectively, our data revealed a requirement for NHEs in vertebrate mucociliary epithelia and linked NHE activity to cilia formation and function in differentiating MCCs. Our results provide an entry point for the understanding of the contribution of NHEs to signaling, development, and pathogenesis in the human respiratory tract.
Project description:This SuperSeries is composed of the following subset Series: GSE22141: MicroRNA signature during the time course of regeneration of the human airway mucociliary epithelium GSE22142: Transcriptome analysis during the time course of regeneration of the human airway mucociliary epithelium GSE22143: Transcriptomic impact of microRNAs-449 or microRNAs-34 overexpression in proliferating human airway epithelial cells GSE22144: miRNAs high throughput sequencing profiling of regenerating human airway epithelial cells GSE22145: miRNAs high throughput sequencing profiling of basals cells and columnar cells GSE22146: microRNAs signatures of Xenopus laevis embryo epidermis at stage 11 (non ciliated) and 26 (ciliated) using high throughput sequencing Refer to individual Series