CHARACTERIZING THE EFFECT OF COLONY STIMULATING FACTORS ON MACROPHAGE METABOLISM AND PHENOTYPE

Abstract

Macrophages are a highly specialized group of immune cells and often referred to as sentinels of the immune system due to their ability to phagocytose cellular debris and pathogens, detect danger signals, and produce cytokines that stimulate or suppress immune responses. Macrophages display remarkable phenotypic plasticity and undergo rapid physiological changes in response microenvironmental stimuli, resulting in heterogenous populations of cells across different tissues. With highly specialized phenotypes that are regulated by a balance of growth factors and pro- and anti-inflammatory cytokines, macrophages are critically important mediators of tissue homeostasis. As a result, macrophage dysregulation is often implicated in the advancement of pathologies such as autoimmune disease and cancer. However, phenotypic distinctions elicited by key growth factors, i.e., GM-CSF and M-CSF, have yet to be fully characterized. Addressing this knowledge gap is important in understanding how macrophage phenotypes are regulated and manipulated across various tissues and disease states. This thesis elucidates how the growth factors GM-CSF and M-CSF impact macrophage differentiation and activation and identifies phenotypic distinctions that further our understanding of macrophage heterogeneity. Additionally, how macrophage subsets respond to immunotherapy, specifically oncolytic reovirus, is explored and characterized. Using quantitative proteomics, among other techniques, phenotypic distinctions between GM-CSF and M-CSF-derived macrophages were first characterized in response to reovirus infection. Here, I show that GM-CSF and M-CSF-derived macrophages have distinct protein expression profiles and differ in their susceptibility to viral infection. Accordingly, M-CSF promotes the higher expression of viral defense proteins whereas GM-CSF promotes the expression of proteins involved in viral entry and endosomal escape. Next, using targeted, untargeted, and stable isotope-tracing metabolomics analysis, I characterized metabolic differences between various macrophage cell-types and identified the kynurenine pathway as an immunometabolic distinction of GM-CSF-stimulated cells. Here, I show the importance of KYNU expression, whereas IDO1 was shown to be enzymatically inactive, allowing the direct catabolism of kynurenine as opposed to tryptophan. Finally, I show that kynurenine pathway enzymes and activity are physiologically relevant in alveolar macrophages, which are also known to be GM-CSF-dependent. All together, this thesis advances our understanding of phenotypic and metabolic distinctions between GM-CSF and M-CSF-derived macrophages, and provides insight on the roles these molecules may have in facilitating macrophage phenotype under steady-state or pathological conditions, improving our overall understanding of growth factor-mediated macrophage differentiation.