A systems based approach to neutrophil gene expression

Thomas, Huw A systems based approach to neutrophil gene expression. PhD thesis, University of Liverpool.

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Abstract

Neutrophils are the major cellular constituent of blood leukocytes and play a central role in the inflammatory response, expressing an array of destructive molecules and antimicrobial processes that characterise the cells as front-line defenders of the innate immune system, thus neutrophils are crucial to host defence. It is now appreciated that neutrophils produce and respond to a variety of inflammatory signals and are able to regulate both the innate and adaptive immune response. The molecular changes that underlie this regulation are poorly defined, yet represent an attractive area of research to fully elucidate the role and regulatory capacity of neutrophils within the immune response. RNA-Seq provides an accurate and robust mechanism for global characterisation of cellular transcripts. Neutrophils were isolated from healthy donors and incubated with or without inflammatory cytokines for 1 h. RNA was extracted and analysed by RNA-Seq using the SOLiD or Illumina platforms. Raw data was quantified using a number of software packages which formed a bioinformatic pipeline for data analysis which was developed during the course of the research. Results were validated by a selection of traditional laboratory functional assays. Priming of neutrophils by GM-CSF and TNFα was found to induce differential gene expression and activation of transcription factors, which led to differential regulation of apoptotic pathways. Stimulation of neutrophils with inflammatory cytokines/chemokines (IL-1β, IL-8, G-CSF, IFNγ) resulted in expression of discrete gene sets and differential activation of signalling pathways. Stimulation of neutrophils with IL-6 did not induce any significant expression of genes but result in activation of STAT signalling. Comparison of gene expression of neutrophils isolated by density gradient and magnetic bead preparation revealed significant differences in gene expression and function, in part attributable to levels of contamination associated with each isolation method. Bead isolation was found to enrich a more heterogeneous neutrophil population including a subpopulation of neutrophils expressing transcripts previously associated with low density granulocytes. Thus, RNA-Seq and bioinformatic analysis has provided a full characterisation of neutrophil gene expression under inflammatory conditions and identified several new areas of research that could lead to targeted drug design for the treatment of inflammatory disease.

Item Type:	Thesis (PhD)
Additional Information:	Date: 2014-09-30 (completed)
Subjects:	?? Q1 ?? ?? QH301 ?? ?? QH426 ??
Depositing User:	Symplectic Admin
Date Deposited:	10 Sep 2015 14:52
Last Modified:	17 Dec 2022 01:05
DOI:	10.17638/02008185
URI:	https://livrepository.liverpool.ac.uk/id/eprint/2008185