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Title: Metabolic programming in murine cytomegalovirus infected macrophages
Author: Kotzamanis, Konstantinos Ioannis
ISNI:       0000 0004 7429 3969
Awarding Body: University of Edinburgh
Current Institution: University of Edinburgh
Date of Award: 2018
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Immunity and metabolism have been viewed as separate fields, however recent evidence show that these two systems are intimately integrated, share resources and cross-regulate each other. Activated immune cells have to alter their metabolism in order to support effector functions. On the other hand, viruses are obligatory parasites that counter and exploit host pathways, including metabolism, to effectively propagate. Like immune cells, viruses have to alter the metabolic profile of infected cells in order to propagate. The regulation of metabolism in immune cells or virally infected cells has been well studied. However, the precise metabolic regulation that ensues when both immune system and viral infection in immune cells interact and compete for the limited resources and metabolic pathways available is not clear. In this thesis, I have sought to investigate the integrative process by studying the metabolic programming of macrophages infected with murine cytomegalovirus (MCMV) The central hypothesis of this thesis is that productive infection of macrophages by MCMV takes advantage of the early inflammatory metabolomic reprogramming of activated macrophages to establish infection, and modulates metabolism at late stages of infection towards fatty acid (FA) production to promote viral progeny. To study this interaction, I have analysed the temporal profile of the transcriptome and metabolome of bone marrow derived macrophages (BMDM) infected with productive (WT) and non-productive (attenuated) (MCMV) strains. This aimed to unravel the host-directed versus virus-driven metabolic alterations. I show evidence indicating that during early times of productive and non-productive MCMV infection glycolysis is, in infected BMDM, markedly increased. Furthermore, pharmacological and siRNA mediated inhibition of glycolysis resulted in attenuation of viral growth demonstrating the dependency of MCMV on this pathway. Additionally, using interferon receptor A (IFNAR) and interferon receptor A (IFNB) deficient BMDM showed that type-I interferon (IFN) signalling is essential for the early upregulation of glycolysis that was observed. In addition to the changes in glycolysis, MCMV infection alters the tricarboxylic acid (TCA) cycle in infected BMDM. Metabolomic and transcriptomic data revealed a shift from catabolic to anabolic function for the TCA to promote production of TCA intermediates. Finally, the urea cycle is also altered both on transcriptional and metabolomic level, consistent with the support of Nitric oxide (NO) production which is a hallmark metabolite in classically activated macrophages. These changes observed in the TCA cycle and glycolysis are consistent with supporting the FA elongation pathway during late time points of productive infection. Only productive MCMV infection upregulates this pathway. At the same time, pharmacological and siRNA mediated inhibition of FA elongation pathway greatly attenuates viral growth. This indicates that MCMV growth is dependenton FA elongation. The effect was very specific for the elongation and not the de novo synthesis pathway indicating that MCMV remodels FA that already in the cells. It is argued, that in agreement to known literature, MCMV uses these FA for the formation of its lipid membrane. To further investigate the dependency of MCMV on FA elongation pathways I studied additional lipids pathway associated with the former. I found that MCMV infection also upregulates the triacylglycerol formation and membrane remodelling pathways, which are dependent on FA biosynthesis and elongation. The inhibition of triacylglycerol formation and membrane remodelling pathway also attenuated MCMV growth. This indicates that apart from the formation of its lipid membrane MCMV requires FA to remodel the cellular environment. I have also explored the effects of infection on regulating lipid mediators, in particular eicosanoids. Eicosanoids are lipid signalling molecules that can act as potent inflammation modulators. Here I demonstrated that productive MCMV infection specifically increases PGE2 production in infected BMDM. Moreover, addition of PGE2 increased viral replication in infected fibroblasts in comparison to non-treated cells, while pharmacological blocking of EP4 (PGE2 receptor) rescued the phenotype. These studies reveal how MCMV advantageously use inflammatory lipid pathways to promote growth In conclusion, the data presented in this thesis support my hypothesis and provide an insight in the role of metabolism during viral infection. Evidence is provided to show that MCMV co-ops the early alterations that metabolic pathways undergo in activated macrophage, including but not limited to glycolysis, TCA cycle and urea cycle. These early changes in metabolism appear to be coupled with upregulation of FA elongation pathways and remodeling of lipids in infected cells. Finally, MCMV co-ops the function of regulatory lipids, in particular PGE2, to promote viral growth. It is further argued that MCMV productive infection dictates these fatty acid metabolism alterations in order to remodel the host cell's environment, regulate the immune system response and provide resources for its lipid membrane.
Supervisor: Ghazal, Peter ; Roy, Douglas Sponsor: Not available
Qualification Name: Thesis (Ph.D.) Qualification Level: Doctoral
EThOS ID:  DOI: Not available
Keywords: metabolism ; macrophages ; immunity ; immunometabolism ; cytomegalovirus