Effect of IL-13 on transcriptome of differentiated human airway mucociliary epithelium
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PROVIDER: PRJNA434581 | ENA |
REPOSITORIES: ENA
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Project description:We used microarrays to detail the global programme of gene expression that occurs in response to treatment of IL-13 on fully-differentiated human airway mucociliary epithelium
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Project description:The regeneration of the airway mucociliary epithelium involves several sequential events including migration, proliferation, polarization and final differentiation (i.e ciliogenesis). We used microarrays to detail the global programme of gene expression that occurs during regeneration and ciliogenesis of the human airway mucociliary epithelium. The four time points of regeneration of the airway epithelium (ALI-D0 which corresponds to the end of proliferation step; ALI-D7 which corresponds to the polarization step; ALI-D14 which corresponds to the onset of ciliogenesis and ALI-D21 corresponding to the terminal differentiation step) for RNA extraction and hybridization on Affymetrix microarrays.
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Project description:Air pollution particulate matter <2.5 microns (PM2.5) is associated with poor respiratory outcomes. Mechanisms underlying PM2.5-induced lung pathobiology are poorly understood, but likely involve cellular and molecular changes to the airway epithelium. We extracted and chemically characterized the organic and water-soluble components of contemporary ambient air pollution PM2.5 samples. We then determined the whole transcriptome responses of human mucociliary airway epithelial cultures (n=12) to a dose series of PM2.5 extracts. We found PM2.5 organic, but not water-soluble, constituents elicited a potent, dose-dependent transcriptomic response from the mucociliary epithelium. Epithelial exposure to a moderate organic extract (OE) dose modified the expression of 424 genes, which included activation of aryl-hydrocarbon receptor (AHR) signaling and an interleukin-1 inflammatory program. We generated an OE response network composed of 8 metagroups, which exhibited high connectivity through the CYP1A1, IL1A, and IL1B genes. This OE exposure also robustly activated a mucus secretory expression program (>100 genes), which included transcriptional drivers of mucus metaplasia (SPDEF, FOXA3). Exposure to a higher OE dose modified the expression of 1,240 genes and further exacerbated expression responses observed at the moderate dose, including the mucus secretory program. Moreover, the higher OE dose significantly increased the MUC5AC/MUC5B gel-forming mucin expression ratio and strongly downregulated ciliated cell expression programs, including key ciliating cell transcription factors (FOXJ1, MCIDAS). Our results suggest organic chemicals in PM2.5 likely drive cellular remodeling of the airway epithelium, punctuated by mucus metaplasia and loss of ciliated cells. This epithelial remodeling may underlie poor respiratory outcomes associated with high PM2.5 exposure.
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Project description:The airways of the human lung are lined by an epithelium made up of ciliated and secretory luminal cells and undifferentiated p63+ Krt5+ basal 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 co-ordinating the expression of numerous proteins required for epithelial morphogenesis, differentiation, remodeling and repair. However, little is known about their function in the adult lung. Here, we focus on the role of GRHL2 in primary human bronchial epithelial (HBE) cells, using either shRNA or a dominant negative protein (DN-GRHL2) to inhibit its function. We follow changes in epithelial phenotype, and in gene transcription using RNA-seq or microarray analysis, both in undifferentiated basal cells and in cells differentiating in air-liquid interface culture into a mucociliary epithelium with transepithelial electrical resistance. We identify several hundreds of genes that are directly or indirectly regulated by GRHL2. Using ChIP-seq 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 GRHL-regulated genes. Different subsets of the large cohort of potential GRHL2 targets appear to be active in basal and differentiated cells. Taken together, the results strongly support the hypothesis that GRHL2 plays a key role in regulating many physiological functions of human airway epithelium, including those involving cell adhesion, polarity and morphogenesis. Frozen primary human bronchial epithelial (HBE) cells were obtained from three donors. Passage 2 cells at 40% confluence were infected with H2B-GFP or DN-GRHL2 lentivirus and 1 mg/ml puromycin added 48 h later. At confluence, Doxycycline 0.5 mg/ml was added for 24 h. RNA-seq was performed on all six samples, as well as samples from two donors that were not infected.
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