Reconstruction of IRG1 Gene Regulatory Network in Mammalian Macrophages under Inflammatory Conditions

The immune system is the first line of defence against invading pathogens. Macrophages are the key effector cells of the innate immune system which produce an array of cytokines, antimicrobial peptides, and effector molecules pathogen in response to pathogen invasion. Immunoresponsive gene 1 (Irg1) is highly upregulated when mouse macrophages are stimulated with LPS. Recently, the function of Irg1 has been elucidated as a gene coding for a protein which catalyses the decarboxylation of cis-aconitate, a tricarboxylic acid (TCA) cycle intermediate, to itaconic acid. In turn, itaconic acid selectively inhibits isocitrate lyase, a key enzyme of the glyoxylate shunt, which is a saviour pathway for bacteria to grow on low carbon diets. Thus, Irg1 via the production of itaconic acid plays an important role in combating pathogen invasion. Despite the importance of this recent discovery, the upstream transcriptional machinery of IRG1 has not yet been investigated. Hence, the aim of this thesis was to elucidate the gene regulatory networks of IRG1 in mammalian macrophages under inflammatory conditions.
To achieve this aim, the experimental protocols for the isolation of monocytes from peripheral blood and their differentiation into macrophages were implemented. The cells were then characterised based on their morphology and the expression profile of cellular marker genes to confirm their identity. Using this cellular model, I discovered IRG1 expression and itaconic acid production in human macrophages under LPS activation. Both IRG1 and itaconic acid were initially discovered elsewhere in murine macrophages upon LPS stimulation. I further analysed the dynamics of IRG1 expression and itaconic acid production using different bacterial and viral ligands in human and mouse macrophages, showing that IRG1 expression could be upregulated when mammalian cells encounter bacteria and viruses.
Owing to the complex upstream transcriptional machinery for IRG1 expression, I implemented a workflow defined as transcription factor identification protocol (TFIP) using both experimental and computational methods to identify potential transcription regulators for IRG1 expression in human and mouse macrophages.
siRNA mediated gene silencing experiments in human and mouse macrophages revealed IRF1 as a transcriptional regulator for IRG1 in both the species and CEBPB in mouse macrophages under LPS activation.