Gene Expression
Nebulas
Gene Expression NebulasSummary: JAK inhibitors dampen activation of interferon-stimulated transcription of ACE2 isoforms in human airway epithelial cells (RNA-seq)
Overall Design: SARS-CoV-2 infection of human airway epithelium activates genetic programs leading to progressive hyperinflammation in COVID-19 patients. Here, we report on transcriptomes activated in primary airway cells by interferons and their suppression by Janus kinase (JAK) inhibitors. Deciphering the regulation of the angiotensin-converting enzyme 2 (ACE2), the receptor for SARS-CoV-2, is paramount for understanding the cell tropism of SARS-CoV-2 infection. ChIP-seq for activating histone marks and Pol II loading identified candidate enhancer elements controlling the ACE2 locus, including the intronic dACE2 promoter. Employing RNA-seq, we demonstrate that interferons activate expression of dACE and, to a lesser extent, the genuine ACE2 gene. Interferon-induced gene expression was mitigated by the JAK inhibitors baricitinib and ruxolitinib used therapeutically in COVID-19 patients. Through integrating RNA-seq and ChIP-seq data we provide an in-depth understanding of genetic programs activated by interferons, and our study highlights JAK inhibitors as suitable tools to suppress these in bronchial cells.
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| Growth Protocol: | Human small airway epithelial cells (SAEC) obtained from Lifeline Technology (FC-0016) were expanded using the complete BronchiaLifeTM media kit (Lifeline Technology, LL-0023). All culture wares were pre-coated in 30 mg/ml of Fibronectin (ThermoFisher Scientific, 33016015) for at least 1h at room temperature. |
| Treatment Protocol: | Cytokines (10 ng/ml; Human IFNb, 300-02BC; Human IFNg, 300-02; Human IL6, 200-06; Human IL7, 200-07; Human Growth hormone, 100-40, Peprotech; Human IFNa2b, 78077.1, Stem Cell Technologies; Human IFNl3, 5259-IL-025, R&D systems) were treated in respective culture media and the cells were incubated for 12hr in 5% CO2 atmosphere at 37°C. Jak inhibitors, 10 mM of either Baricitinib (HY-15315A, MedChemExpress) or Ruxolitinib (HY-50856A, MedChemExpress), were added to BronchiLiafeTM media with or without IFNb and incubated for 12hrs. |
| Extract Protocol: | Total RNA was isolated from the cytokine-stimulated cells using PureLink™ RNA Mini Kit (Invitrogen) and libraries for sequencing were prepared according to the manufacturer’s instructions with TruSeq Stranded Total RNA Library Prep Kit with Ribo-Zero Gold (Illumina, RS-122-2301). Libraries for sequencing were prepared using standard Illumina protocols. |
| Library Construction Protocol: | Libraries for sequencing were prepared according to the manufacturer’s instructions with TruSeq Stranded Total RNA Library Prep Kit with Ribo-Zero Gold (Illumina, RS-122–2301) |
| Molecule Type: | rRNA- RNA |
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| Library Layout: | PAIRED |
| Library Strand: | Forward |
| Platform: | ILLUMINA |
| Instrument Model: | Illumina HiSeq 3000 |
| Strand-Specific: | Specific |