Investigating the contribution of Immunoresponsive gene 1 (Irg1) to macrophage functions and its relevance to disease

Abstract

Macrophages are essential effectors in the innate immune responses to injury, infection, and disease. Their broad functional plasticity allows them to tailor their behaviours to the specific homeostatic or immune needs of their microenvironment. However, macrophages have also been connected to the propagation of undesirable inflammation and associated disease, as in the case of obesity or neurodegeneration. An understanding of the drivers that determine macrophage phenotype is essential for the interpretation of macrophage roles in different immune environments and to inform how the process of phenotype switching may be modulated for therapeutic benefit. The growing field of immunometabolism proposes that functional changes within macrophages are derived from shifts in metabolic preference. This thesis presents the infection-responsive, mitochondrial enzyme Immunoresponsive gene 1 (Irg1) as a metabolic driver of macrophage activation and effector functions. Generation of Tg(irg1:EGFP)nz4, a unique transgenic zebrafish reporter line that marks expression of irg1 in macrophage-lineage cells, has enabled, for the first time, observation of the in vivo process of macrophage activation. This thesis has further exploited the advantages of the Tg(irg1:EGFP)nz4 transgenic line for transcriptome analysis of activated macrophages responding to LPS with or without the contribution of functional Irg1. This has led to the discovery that Irg1 exerts its effector functions in part by modulating NFκB signalling pathways and inducing the expression of potent inflammatory cytokines, including TNFα and IL-1β. Irg1 is anticipated to mediate these effects through mROS produced by Irg1-driven fatty acid β oxidation, drawing further support for a connection between metabolism and immunity in inflammatory disease. Finally, a novel aqueous screening platform has been developed to facilitate a drug-repurposing screen aimed at identifying modulators of irg1 expression. This screen has revealed a number of strong candidates for the therapeutic reduction of irg1 expression. In sum, the Tg(irg1:EGFP)nz4 transgenic has provided invaluable insight into the process of macrophage activation through investigations into the downstream consequences of Irg1 activity. This transgenic line is anticipated to be of great value for further analyses of macrophage plasticity and effector functions. In addition, small molecules have been identified that may have applications to the therapeutic modulation of Irg1 activity in instances where macrophage-driven inflammation is associated with the propagation of disease. Together, this thesis has advanced understanding of the regulation of inflammatory effector functions in macrophage-lineage cells through Irg1.