Using single-cell RNA sequencing to investigate inflammatory responses in human physiology and pathology

Abstract

Transcriptomic analysis can provide valuable insight into the functional and dynamic elements of the genome. Traditional bulk transcriptomic methodologies measure an averaged expression level for each gene across a population of cells. In doing so, the stochastic nature of gene expression is ignored and vital heterogeneity within a diverse cell population is masked. The emergence of single-cell RNA sequencing (scRNA-seq) has addressed such issues by probing the transcriptomic landscape of multiple individual cells simultaneously. The unprecedented multi-dimensional data yielded by scRNA-seq analysis has helped uncover gene expression patterns to answer complex biological research questions.

In this thesis we utilised the potential of scRNA-seq to explore a number of objectives concerning inflammation in human health and disease. Comparing the gene expression profiles of synovial fibroblasts isolated from the hip joint of osteoarthritis normal-weight and obese patients, we uncovered divergent signalling mechanisms with prominent markers of inflammation in cells obtained from obese patients. Also in OA pathology, we gained insight into the mechanisms responsible for pain mediation by comparing the expression profiles of synovial fibroblasts isolated from the knee joints of painful compared to non-painful areas. In this study we discovered specific subsets of cells from the painful areas, associated with high expression of pro-inflammatory markers, compared to non-painful areas showing neurite growth potential. Furthermore, we also explored changes in gene expression dynamics to model the transition of pain from early to end-stage OA disease; uncovering vital signalling mechanisms such as eicosanoid and prostanoid mechanisms in end-stage disease.

In ankylosing spondylitis (AS) and psoriatic arthritis (PsA) we used scRNA-seq to explore the role of individual CD45+ peripheral blood mononuclear cell types in propagating systemic inflammation. Significantly, this study highlighted the role of IFN-힬-mediated signalling in PsA pathology, compared to AS. We also identified specific differential expression of the transcripts HLA-DQA2 and AL365357.1 in the PsA-isolated cells, that could potentially serve as biomarkers of disease.

Dexamethasone (Dex) is a potent, synthetic glucocorticoid drug that is used as an anti-inflammatory and immunosuppressant agent, to treat a range of inflammatory conditions including AS, PsA and OA. Despite its wide use in the clinic, the exact mechanism-of-action is not fully understood. Thus, in the final chapter of this thesis, we used scRNA-seq to explore the mechanism of action of dexamethasone in lipopolysaccharide-stimulated THP-1 monocyte cells. From this study, we were able to uncover novel genes such as ONECUT2, SLIRP and IL27RA that may be involved in delivering anti-inflammatory relief by dexamethasone.

Date of Award	21 Jul 2021
Original language	English
Awarding Institution	University of Bath
Supervisor	Mark Lindsay (Supervisor) & Neil McHugh (Supervisor)