Project description:The purpose of the study is to compare the transcriptomic profile of the airway epithelial cell line Calu-3 in reponse to Pseudomonas aeruginosa virulence factor flagellin. CF is caused by mutations of the cystic fibrosis transmembrane conductance regulator (CFTR) gene. CFTR gene in Calu-3 cells was knockdown by CRISPR-Cas9 (TS) and a control cell line (CTL) was aslo generated using a non-targeting guide RNA. CTL and CFTR KD Calu3-cells were grown on Transwell filters, polarized at the air-liquid interface and exposed to flagellin for 6 hours. We considered 2 non-stimulated (ND) conditions: CTL-NS and TS-NS, and 2 conditions stimulated with flagellin (Flag): CTL-Flag and TS-Flag.
Project description:Here, we aim to understand the role of P38 in the airway epithelial cells response to flagellin. We report the gene expression profile of pig airway epithelial cells cultured under air-liquid conditions, pre-incubated or not with the P38 inhibitor SB203580 (20 µM, Tocris Bioscience) for 1 h and then stimulated with 100 ng/ml of a mutated flagellin (FliCΔ174-400) for 2h or 24h. Our data show that flagellin stimulation induce the expression of pro-inflammatory cytokines mainly through the NFkB pathway, with little impact of P38 inhibition in the cells response.
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.
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.
Project description:Ets homologous factor (EHF) is an Ets family transcription factor expressed in many epithelial cell types including those lining the respiratory system. Disruption of the airway epithelium is central to many lung diseases, and a network of transcription factors coordinates its normal function. EHF can act as a transcriptional activator or a repressor, though its targets in lung epithelial cells are largely uncharacterized. Chromatin immunoprecipitation followed by deep sequencing (ChIP-seq), showed that the majority of EHF binding sites in lung epithelial cells are intergenic or intronic and coincide with putative enhancers, marked by specific histone modifications and increased DNase hypersensitivity. Examination of open chromatin region in Calu-3 cells with 1 replicate
Project description:This series contains 5 groups of U133A arrays with targets from Calu-3 cell line post-infection with P aeruginosa strains. Calu-3 human lung epithelial cells were seeded onto 6-well plates and grown to about 80% confluency. Epithelial monolayers were infected with 108 CFUs per ml of each bacterial strain for 60 min and then washed extensively with phosphate-buffered saline. The cultures were subsequently incubated in fresh RPMI 1640 medium supplemented with 10% fetal bovine serum and 100 µg/ml gentamicin. After 6 h, total RNA from the cells was prepared with RNAzol and further purified with RNEasy for microarray hybridization. All arrays processed and globally scaled to 500 using MAS 5. CONTROL (4 arrays) - no infection FRD1 (4 arrays) FRD1234 (3 arrays) FRD440 (4 arrays) FRD875 (4 arrays) Keywords = Cystic Fibrosis Keywords = Pseudomonas aeruginosa Keywords = CF Keywords = flagellin Keywords = alginate Keywords = lung Keywords: repeat sample
Project description:Ets homologous factor (EHF) is an Ets family transcription factor expressed in many epithelial cell types including those lining the respiratory system. Disruption of the airway epithelium is central to many lung diseases, and a network of transcription factors coordinates its normal function. EHF can act as a transcriptional activator or a repressor, though its targets in lung epithelial cells are largely uncharacterized. RNA-seq after EHF depletion or overexpression showed significant alterations in the expression of genes involved in response to wounding. EHF knockdown also targeted genes in pathways of epithelial development and differentiation and locomotory behavior. mRNA profiles from Calu-3 cells treated with negative control (NC) or EHF siRNA, in quintuplicate. mRNA profiles from 3 pcDNA (empty vector control) clones and 3 pcDNA-EHF overexpression A549 clones, 3-4 replicates each.
Project description:Ets homologous factor (EHF) is an Ets family transcription factor expressed in many epithelial cell types including those lining the respiratory system. Disruption of the airway epithelium is central to many lung diseases, and a network of transcription factors coordinates its normal function. EHF can act as a transcriptional activator or a repressor, though its targets in lung epithelial cells are largely uncharacterized. Chromatin immunoprecipitation followed by deep sequencing (ChIP-seq), showed that the majority of EHF binding sites in lung epithelial cells are intergenic or intronic and coincide with putative enhancers, marked by specific histone modifications. EHF occupies many genomic sites that are close to genes involved in intercellular and cell-matrix adhesion. Examination of EHF binding plus two histone modifications in Calu-3 cells. There are two replicates of the EHF ChIP-seq plus an input sample (control). There is one replicate each of the two histone modifications, plus a second input sample (control).
Project description:Analysis of gene expression in Caco-2 intestinal epithelial cells stimulated with flagellated or aflagellated S. enteritidis or recombinant flagellin. Results provide insight into how flagellin mediates innate immune responses in intestinal epithelial cells. Keywords: Gene expression analysis
Project description:SARS-CoV-2, the virus responsible for COVID-19, infects both human airway epithelial cells and trigeminal ganglia. We assessed the consequences of SARS-CoV-2 infection on gene expression in Calu-3 cells and in primary Mus musculus trigeminal ganglia cells (mmTG). Here, we provide datasets that include raw reads and mapped reads for the following: 1) mock infected mmTG 48 hrs post infection; 2) SARS-CoV-2 infected mmTG 48 hrs post infection; 3) mock infected Calu-3 cells 24 hrs post infection; 4) SARS-CoV-2 infected Calu-3 cells 24 hrs post infection; 5) mock infected Calu-3 cells 48 hrs post infection; 6) SARS-CoV-2 infected Calu-3 cells 48 hrs post infection.