A systems based approach to studying neutrophil gene expression

It is now appreciated that neutrophils play key roles in driving inflammation and tissue damage (via release of proteases) in inflammatory diseases. These cells would thus appear to be an attractive target for therapy, but before this can be realised, greater knowledge of the basic biology of activated neutrophils is required. It is also recognised that activated neutrophils possess most, if not all, of the molecular properties that were once thought to be restricted to macrophages. Thus, activated neutrophils (but not bloodstream neutrophils) can present antigen to T cells via expression of class II MHC, and can express and secrete cytokines and chemokines that can orchestrate immune function in inflammation. Our work has shown that signalling pathways involving hypoxia, FcgRIIIb and cytokines are intimately involved in regulating neutrophil function. The aims of this project are to provide a systems-based approach to define the basic functional properties of inflammatory neutrophils, as the first step towards the identification of specific pathways that could serve as future targets for rational drug design. Previous work in our laboratories has identified FcgRIIIb, an isoform of CD16 uniquely expressed on neutrophils, as a potential therapeutic target, for the following reasons: (i) its experimental blocking or removal completely prevents the release of proteases and reactive oxidants from cytokine-treated neutrophils in response to soluble immune complexes (of the type found in auto-immune diseases); blocking the function of FcgRIIIb had absolutely no effect on the phagocytosis and killing of serum-opsonised Staphylococcus aureus; (iii) individuals with a gene deficiency of FcgRIIIb are devoid of this receptor on their neutrophils and yet are asymptomatic and have no increased susceptibility to bacterial or fungal infections. Thus, FcgRIIIb may be a highly selective drug target: blocking its function should prevent host tissue damage in response to soluble immune complexes, but not compromise host defence against infections. Recent advances in high throughput sequencing technologies have revolutionalised the costs and capacity of genome analyses. Moreover, the introduction of 'digital transcriptomics' whereby cDNA libraries from cells and tissues are rapidly sequenced and analysed in a fraction of the time and for a fraction of the cost of Sanger based sequencing strategies, offers considerable advances over micro-array based studies of the transcriptome. For example, digital transcriptomics: is quantitative; does not depend on prior micro-array design; can quantify changes in exon usage and can detect and quantify SNPs and other polymorphisms. Recent advances in bioinformatics and computational approaches to data analyses can generate predictive models of the signalling pathways and molecular phenotypes of cells, based on such transcriptome studies. This project will study the basic biology of neutrophils activated in vitro under conditions that occur during inflammation. We have identified that cytokines, acidosis, hypoxia and FcgRIIIb signalling all regulate neutrophil function during inflammation and induce functional changes via altered gene expression. This project will thus employ digital transcriptomics to examine the effects of these parameters on neutrophil gene expression, either in isolation or in combination (e.g. cytokines + acidosis; Hypoxia + acidosis; FcgRIIIb ligation + hypoxia+ acidosis, etc). Full bioinformatics analysis of this data will lead to the development of a predictive model to determine the functional consequences of these changes in gene expression. This could then be used to predict, and ultimately measure, altered neutrophil function(s) in vivo, and ultimately new target identification. The project will thus provide the student with first rate training in a range of post-genomic and quantitative skills, that will be useful for a future career in either academia or industry.