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Gene expression analysis of airway epithelial cells exposed to flagellin via RNA-seq

ABSTRACT: Airway epithelial cells (AEC) are critical components of the inflammatory and immune response during exposure to pathogens. AECs in monolayer culture and differentiated epithelial cells in air-liquid interface (ALI) represent two distinct and commonly used in vitro models, yet differences in their response to pathogens have not been investigated. In this study, we compared the transcriptional effects of flagellin on AECs in monolayer culture versus ALI culture using exon microarrays and RNAsequencing. We found that AECs cultured in monolayer and ALI have strikingly different transcriptional states at baseline. When challenged with flagellin, monolayer AEC cultures greatly increased transcription of numerous genes mapping to wounding response, immunity and inflammatory response. In contrast, AECs in ALI culture had an unexpectedly muted response to flagellin, both in number of genes expressed and relative enrichment of inflammatory and immune pathways. In conclusion, In vitro culturing methods have a dramatic effect on the transcriptional profile of AECs at baseline and after stimulation with flagellin. These differences suggest that epithelial responses to pathogen challenges are distinctly different in culture models of intact and injured epithelium.

ORGANISM(S): Homo sapiens

PROVIDER: GSE55458 | GEO | 2014/12/01

SECONDARY ACCESSION(S): PRJNA239685

REPOSITORIES: GEO

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Gene expression analysis of airway epithelial cells exposed to flagellin via microarray

Project description:Airway epithelial cells (AEC) are critical components of the inflammatory and immune response during exposure to pathogens. AECs in monolayer culture and differentiated epithelial cells in air-liquid interface (ALI) represent two distinct and commonly used in vitro models, yet differences in their response to pathogens have not been investigated. In this study, we compared the transcriptional effects of flagellin on AECs in monolayer culture versus ALI culture using exon microarrays and RNAsequencing. We found that AECs cultured in monolayer and ALI have strikingly different transcriptional states at baseline. When challenged with flagellin, monolayer AEC cultures greatly increased transcription of numerous genes mapping to wounding response, immunity and inflammatory response. In contrast, AECs in ALI culture had an unexpectedly muted response to flagellin, both in number of genes expressed and relative enrichment of inflammatory and immune pathways. In conclusion, In vitro culturing methods have a dramatic effect on the transcriptional profile of AECs at baseline and after stimulation with flagellin. These differences suggest that epithelial responses to pathogen challenges are distinctly different in culture models of intact and injured epithelium.

2014-12-01 | GSE55459 | GEO

Development of novel in vitro human alveolar epithelial cell models to study distal lung biology and disease [scRNA-seq]

Project description:To address the need for human alveolar epithelial cell (AEC)-derived lines to more suitably model distal lung diseases, we have generated and characterized novel immortalized cell lines derived from human AECs. We used a combination of the ROCK inhibitor, Y-27632, and lentiviral transduction of SV40 Large T antigen of previously cryopreserved isolated human alveolar epithelial type 2 (AT2) cells to generate immortalized AECs. These AEC lines proliferate well on standard tissue culture dishes forming an epithelial monolayer and express lung progenitor markers SOX9 and SOX2. When grown in 3D culture with lung fibroblasts, the cells form NKX2-1+ organoids expressing more mature alveolar lung markers, AQP5 and GPRC5A. Single cell RNA-sequencing of one AEC line comparing cells in 2D versus 3D revealed increased cellular heterogeneity and an induction of cytokine and lipoprotein signaling in 3D culture, reflecting interactions with the microenvironment during organoid formation. Taken together, these data show our novel progenitor-like AEC lines retain a genetic and structural memory of their alveolar cell lineage despite long-term expansion, providing a valuable new system to model the distal lung in vitro.

2022-01-10 | GSE164514 | GEO

Development of novel in vitro human alveolar epithelial cell models to study distal lung biology and disease [bulk RNA-seq]

2022-01-10 | GSE164513 | GEO

DNA Methylation Profiles of Airway Epithelial Cells and PBMCs from Healthy, Atopic and Asthmatic Children.

Project description:DNA methylation profiling of airway epithelial cells (AECs) and peripheral blood mononuclear cells (PBMCs) from normal, atopic and asthmatic subjects. The Illumina GoldenGate Methylation Cancer Panel I was used to obtain DNA methylation profiles across approximately 1505 CpGs in AECs and PBMCs. Samples included 7 healthy, 9 atopic, 4 atopic asthmatic and 5 non-atopic asthmatic subjects. Please note that only some of the samples are matched (i.e. AECs and PBMCs from the same individual) due to DNA quality or sample collection (i.e. only one sample (AEC or PBMC) was collected from the patient). Bisulphite converted DNA from the 41 samples were hybridised to the Illumina GoldenGate Methylation Beadchip

2012-05-08 | E-GEOD-37853 | biostudies-arrayexpress

RHINOVIRUS INFECTION OF THE AIRWAY EPITHELIUM ENHANCES MAST CELL IMMUNE RESPONSES VIA EPITHELIAL-DERIVED INTERFERONS

Project description:Rationale: Mast cells (MCs) within the airway epithelium in asthma are closely related to airway dysfunction, but crosstalk between airway epithelial cells (AECs) and MCs in asthma remains incompletely understood. Human rhinovirus (HRV) infections are key triggers for asthma progression and AECs from individuals with asthma may have dysregulated anti-viral responses. Objectives: We utilize primary phenotyped AECs in an ex vivo coculture model system to examine crosstalk between AECs and MCs following epithelial HRV infection. Methods: Primary AECs were obtained from children with asthma (n=11) and healthy children (n=10), differentiated at air-liquid interface, and cultured in the presence of laboratory of allergic diseases-2 (LAD2) MCs. AECs were infected with HRV serogroup A 16 (HRV16) for 48 hours. RNA isolated from both AECs and MCs underwent RNA-sequencing (RNAseq) analysis. Direct effects of epithelial-derived interferons on LAD2 MCs were examined by qPCR. Results: MCs increased expression of pro-inflammatory and anti-viral genes in AECs. AECs demonstrated a robust antiviral response following HRV16 infection that resulted in significant changes in MC gene expression, including upregulation of genes involved in anti-viral responses, leukocyte activation, and type-2 (T2) inflammation. Subsequent ex vivo modeling demonstrated that IFN-β induces MC IL13 expression. The effects of AEC phenotype were small relative to the effects of viral infection and the presence of MCs. Conclusions: There is significant crosstalk between AECs and MCs, which are present in the epithelium in asthma. Epithelial-derived interferons not only play a role in viral suppression, but further alter MC immune responses including specific T2 genes.

2022-06-22 | GSE206680 | GEO

Changes in microRNA and mRNA expression with differentiation of human bronchial epithelial cells [mRNA]

Project description:Normal human bronchial epithelial (NHBE) cells cultured in an air-liquid interface (ALI) system form a polarized, pseudostratified epithelium composed of basal, ciliated and goblet cells that closely resemble the in vivo airway epithelium structure. ALI cultures of NHBE cells provide a unique in vitro system to investigate airway epithelial biology, including developmental, structural and physiologic aspects. In this study, we wanted to investigate mRNA expression patterns during airway epithelium differentiation. By using microarrays, we studied the changes in expression of mRNAs in normal human bronchial epithelial cells as they differentiate from an undifferentiated monolayer to a differentiated pseudostratified epithelium after 28 days of air-liquid interface (ALI) culture, when epithelial cells differentially express basal, ciliated and goblet cell markers. Normal human bronchial epithelial cells were cultured in an air-liquid interface (ALI) system and harvested at three different time-points: subconfluent, confluent and day 28 of ALI. Samples were processed for total RNA extraction and hybridization on Affymetrix microarrays. All the experiments were performed by triplicate.

2012-10-01 | E-GEOD-39059 | biostudies-arrayexpress

Transcription profiling of fetal lung from wild type and CREB knock-out mice

Project description:The cAMP response element binding protein (Creb) is a member of a leucine zipper transcription factor family that regulates gene expression primarily in response to the intracellular cAMP signalling pathway. Previous studies have shown Creb1-null mice suffer respiratory failure with lung atelectasis and a large reduction in Sftpd mRNA. Using a new line of Creb1-null mice we have further investigated Creb function in the developing mouse lung, focussing on differentiation of the airway epithelium. The lungs of Creb1-null fetal mice showed normal respiratory development until E17.5 when proximal and distal airways fail to inflate. Subsequent ultrastructural analysis of the lungs of E17.5 Creb1-null fetal mice revealed a defect in AEC differentiation with a reduction in proportions of type-II AECs and in particular, a very large reduction in type-I AECs. Furthermore, immunostaining for the proximal epithelial cell markers Scgb1a1 (also known as CC10) (Clara cells), Foxj1 (Ciliated cells), and CGRP (Neuroendocrine cells) showed delayed or defective proximal epithelial differentiation in Creb1-null fetal lungs. Quantitative real time PCR (qRT-PCR) analysis at E17.5 in Creb1-null fetal lungs showed differential expression of mRNAs for Creb/Atf1 subfamily members, surfactant-associated proteins, type-I AEC markers and proximal epithelial markers. Furthermore, whole-genome microarray analysis at E17.5 in Creb1-null fetal lungs has provided novel genes which will prove useful to further investigate Creb-mediated signalling in lung development. Together these results demonstrate that Creb plays a key role in determining cell lineages and differentiation of the developing lung epithelium.

2009-04-30 | E-MEXP-1295 | biostudies-arrayexpress

DNA Methylation Profiles of Airway Epithelial Cells and PBMCs from Healthy, Atopic and Asthmatic Children.

2012-05-09 | GSE37853 | GEO

Changes in microRNA and mRNA expression with differentiation of human bronchial epithelial cells [mRNA]

2012-10-01 | GSE39059 | GEO

Changes in microRNA and mRNA expression with differentiation of human bronchial epithelial cells [miRNA]

Project description:Normal human bronchial epithelial (NHBE) cells cultured in an air-liquid interface (ALI) system form a polarized, pseudostratified epithelium composed of basal, ciliated and goblet cells that closely resemble the in vivo airway epithelium structure. ALI cultures of NHBE cells provide a unique in vitro system to investigate airway epithelial biology, including developmental, structural and physiologic aspects. MicroRNAs (miRNAs) are short, single-stranded, non-coding RNAs of 20-23 nucleotides that down-regulate gene expression by either inducing degradation of target mRNAs or impairing their translation. They are phylogenetically well conserved, which probably implies an important role of miRNAs in biological processes. In this way, we wanted to shed some light on miRNA specific roles and the relationship with their mRNA targets during airway epithelium differentiation. By using microarrays, we studied the changes in expression of microRNAs in normal human bronchial epithelial cells as they differentiate from an undifferentiated monolayer to a differentiated pseudostratified epithelium after 28 days of air-liquid interface (ALI) culture, when epithelial cells differentially express basal, ciliated and goblet cell markers. Normal human bronchial epithelial cells were cultured in an air-liquid interface (ALI) system and harvested at three different time-points: subconfluent, confluent and day 28 of ALI. Samples were processed for total RNA (including small RNAs) extraction and hybridization on Affymetrix microarrays. All the experiments were performed by triplicate.

2012-10-01 | E-GEOD-39060 | biostudies-arrayexpress

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