Progression of whole blood transcriptional signatures from interferon-induced to neutrophil-associated patterns in patients with severe influenza

Transcriptional profiles are increasingly used to investigate the severity, subtype and pathogenesis of disease. We now describe whole blood RNA signatures and local and systemic immune mediator levels in a large cohort of adults hospitalised with influenza from which extensive clinical and investigational data was obtained. Signatures reflecting interferon-related antiviral pathways were common up to day 4 of symptoms in cases not requiring mechanical ventilatory support; in those needing mechanical ventilation, an inflammatory, activated neutrophil and cell stress/death (‘bacterial’) pattern was seen, even early after disease onset. Identifiable bacterial co-infection was not necessary for this ‘bacterial’ signature but could enhance its development while attenuating the early ‘viral’ signature. Our findings emphasise the importance of timing and severity in the interpretation of transcriptomic profiles and soluble mediator levels, and identify specific patterns of immune activation that may enable the development of novel diagnostics and therapeutics At each time point, 3 ml of whole blood were collected into each of two Tempus tubes (Applied Biosystems/Ambion) by trained research staff following a standard phlebotomy protocol. Blood was vigorously mixed immediately following collection and stored at -80°C before RNA extraction. For each patient, the contents of one tube were used for analysis and the other tube was retained in case of assay failure. RNA was isolated using 1.5 ml whole blood and the MagMAX-96 Blood RNA Isolation Kit (Applied Biosystems/Ambion), as per the manufacturer’s instructions. 250 ug of isolated total RNA was globin-reduced using the GLOBINclear 96-well format kit (Applied Biosystems/Ambion) according to the manufacturer’s instructions. Total and globin-reduced RNA integrity was assessed using an Agilent 2100 Bioanalyzer (Agilent Technologies). RNA yield was assessed using a NanoDrop8000 spectrophotometer (NanoDrop Products, Thermo Fisher Scientific). High-quality (>6.5 RIN) whole blood RNA was successfully obtained and processed by microarray in all cases. Biotinylated, amplified antisense complementary RNA (cRNA) targets were prepared from 200-250 ng of globin-reduced RNA using the Illumina CustomPrep RNA amplification kit (Applied Biosystems/Ambion). For each sample, seven hundred and fifty nanograms of labelled cRNA were hybridised overnight to Illumina Human HT12 V4 BeadChip arrays (Illumina), which contained greater than 47,000 probes. The arrays were washed, blocked, stained and scanned on an Illumina iScan, as per the manufacturer’s instructions. GenomeStudio (Illumina) was used to perform quality control and generate signal intensity values. Contributor: the MOSAIC Investigators