Endogenous plasma resuspension of peripheral blood mononuclear cells prevents preparative-associated stress that modifies polyA-enriched RNA responses to subsequent acute stressors

Authors:

Dongyang Li1,2, Karina Al-Dahleh1,2, Daniel A Murphy2,3, Sonya Georgieva1, Nik Matthews4 and Claire L Shovlin1,2,5,*
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Affiliations:

1 National Heart and Lung Institute, Imperial College London, W12 ONN, London, UK. 2 National Institute for Health Research (NIHR) Imperial Biomedical Research Centre, W2 1NY, London, UK. 3 Pharmacy, Imperial College Healthcare NHS Trust, W12 OHS, London, UK. 4 NIHR Imperial BRC Genomic Facility, Faculty of Medicine, Imperial College London. 5 Specialist Medicine, Imperial College Healthcare NHS Trust, W12 OHS, London, UK.

Keywords: 

Cycloheximide, hypothermia, integrated stress response, intronless (single exon) genes, iron, protein translation inhibition, reactive oxygen species, poly-A selected RNASeq, experimental variability.

Abbreviations:

ATF - activating transcription factor,

eiF2 - eukaryotic initiation factor 2,

GO - gene ontology,

NMD - nonsense-mediated decay,

PBMC - peripheral blood mononuclear cell,

TNF - tumor necrosis factor.

Corresponding Author(s):

Claire L Shovlin, PhD FRCP, Professor of Practice (Clinical and Molecular Medicine), National Heart and Lung Institute, Imperial College London, Hammersmith Campus, Du Cane Road, London W12 0NN, UK.; c.shovlin@imperial.ac.uk

Conflict of interest statement:

The authors have no direct conflict of interest to declare. The use of MEK inhibitors for treatment of bleeding in hereditary haemorrhagic telangiectasia (HHT) is the subject of a patent application by Imperial College London.

Please cite this article as:

Dongyang Li, Karina Al-Dahleh, Daniel A Murphy, Sonya Georgieva, Nik Matthews, Claire L Shovlin (2024). Endogenous plasma resuspension of peripheral blood mononuclear cells prevents preparative-associated stress that modifies polyA-enriched RNA responses to subsequent acute stressors. Cell Stress 11: 112-124. doi: 10.15698/cst2024.11.301

© 2024 Li et al. This is an open-access article released under the terms of the Creative Commons Attribution (CC BY) license, which allows the unrestricted use, distribution, and reproduction in any medium, provided the original author and source are acknowledged.

Abstract:

Human peripheral blood mononuclear cells (PBMCs) are used to examine biological processes and disease, when basal variability in cellular activation and splicing is described and unexplained. Using isolation systems that maintained buffy coat cells (PBMCs, platelets) in their own plasma, poly-A enriched RNA-sequencing (RNASeq) detected 42,720 Ensembl gene IDs, including >95% of the top 100 Genotype Tissue Expression Project (GTEx)-expressed genes in lung, colon, heart, skeletal muscle and liver, and 10/17 clinically-actionable genes listed by the Pharmacogenomics Knowledgebase. Transcriptome changes were defined after 1h treatment with 32◦C hypothermia (hsp70 family member change), 10 µmol/L ferric citrate that had no discernible effect, and 100 µg/mL cycloheximide leading to induction of primary response (immediate early) genes including IL1B and TNF. Same-donor PBMCs prepared conventionally using washes then resuspension in serum-supplemented media demonstrated basal upregulation of stress signalling pathway genes that masked and overlapped differential gene  expression profiles after 100 µg/L cycloheximide. Plasma-resuspended PBMCs demonstrated minor transcriptome changes after 40 µmol/L ferric citrate, whereas consistent and greater magnitude changes were observed for washed/media- resuspended PBMCs. We conclude that endogenous plasma-maintained PBMCs provide a more robust platform to interrogate acute cellular perturbations trig- gering innate immunity, and that varying susceptibility of PBMCs to preparative stresses is an important component of experimental variability.