Host metabolism is profoundly altered during bacterial infection, both as a consequence of immune activation and secondary to virulence strategies of invading pathogens. As a result, the metabolic pathways that regulate nutrient acquisition, energy storage, and resource allocation in host cells must adapt to pathogen stress in order to meet the physiological demands of the host during infection. In this work, we uncover that the synthesis of the monounsaturated fatty acid (MUFA) oleate is necessary for the pathogen-mediated induction of immune defense genes. Accordingly, C. elegans deficient in oleate production are hypersusceptible to infection with diverse human pathogens, which can be rescued by the addition of exogenous oleate. However, oleate is not sufficient to drive protective immune activation. Oleate is also important for proper lipid storage and abundance. We found that exposure to pathogenic bacteria drives rapid somatic depletion of lipid stores in C. elegans. Activating the p38/MAPK immune signaling pathway in the absence of pathogens was also sufficient to drive loss of somatic fat. In addition, we found that transcriptional suppression of MUFA synthesis occurs during P. aeruginosa infection, in a manner dependent on pathogen virulence. Finally, we showed that the host compensates for the pathogen-induced depletion of fatty acids by promoting the redistribution of oleate from non-intestinal tissues to support immune function in the intestine. Together, these data add to the known health-promoting effects of MUFAs, and suggest an ancient link between nutrient stores, metabolism, and host responses to bacterial infection.

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Liver injury instigates a proinflammatory response in tissue-resident macrophages, called Kupffer cells (KCs), resulting in the recruitment of monocytes and neutrophils. The high energy demand required for a rapid proinflammatory response in macrophages like KCs is achieved through metabolic reprogramming. This is supported by increased glycolysis. On the other hand, injury resolution requires hepatic macrophages to undergo an anti-inflammatory polarization, which relies on oxidative phosphorylation (OXPHOS). In addition to shifts in mechanisms of adenosine triphosphate (ATP) production, lipid metabolic reprogramming supplies metabolic intermediates and lipids for membrane remodeling and the production of inflammatory mediators. AMP-activated protein kinase (AMPK) is a master metabolic regulator that influences the metabolic reprogramming of macrophages. While AMPK activation promotes an anti-inflammatory polarization, disruption of activity exacerbates proinflammatory signaling. For this thesis work, we addressed whether macrophage AMPK is protective against liver injury by altering immunometabolism. Specifically, we investigated this question in the context of chronic (nonalcoholic steatohepatitis (NASH)) and acute (acetaminophen (APAP) overdose) liver injury. While APAP overdose is a robust and directly translational model of acute injury, models of NASH-induced hepatic fibrosis rely on nutrient-deficient diets like the choline-deficient high-fat diet (CDAHFD) or genetic manipulation. Despite the utility of these models, they seldom mirror the pathogenesis of human NASH, with diets like CDAHFD being completely dissociated from metabolic syndrome. Moreover, models are required to address the divergence between male and female mice. Recently, there has been a shift towards addressing other variables that drive inflammation and metabolism. At room temperature (RT) (22 °C), mice experience cold stress that alters various biological functions. Cold stress drives brown adipose tissue (BAT) activation and upregulates corticosterone production and immunosuppression, all processes that blunt NASH progression. Giles et al. (2016) demonstrated that housing mice at thermoneutrality (TN) (30 °C) exacerbated metabolic-dysfunction associated fatty liver disease (MAFLD) progression toward NASH in both male and female mice. Since then, we and others have implemented TN housing with different dietary interventions and mice strains. We determined that 16-week Western diet (WD) feeding of male and female mice at 29 °C was insufficient to drive hepatic fibrosis, however alterations in glucose tolerance and elevated liver injury enzymes as well as profibrotic gene expression in male mice may indicate that a longer timeline is necessary (24 weeks). Given that our TN NASH model did not produce hepatic fibrosis, we implemented the CDAHFD to investigate macrophage AMPK in chronic liver injury. Male and female AMPK Flox (Prkaa1 fl/fl/Prkaa2 fl/fl) and MacKO (Flox-LysM-Cre+) mice were fed CDAHFD for 8 weeks. In this time frame, CDAHFD produces a lean euglycemic phenotype with hepatic steatosis, inflammation, and fibrosis, to which AMPK MacKO had no influence. Moreover, intervention with a low dose of metformin had no effect, contrary to the reduction in hepatic steatosis observed in HFD-fed mice. Although macrophage AMPK is dispensable in the CDAHFD model of chronic liver injury, acute liver injury needed to be addressed. We found that priming with systemic activation of a direct AMPK activator MK-8722 did not influence hepatic injury and necrosis in our model of APAP-induced liver injury (AILI). Moreover, deletion of hepatocellular AMPK (Flox-Alb-Cre+) or AMPK MacKO did not influence injury at 24 hours post overdose. Despite the lack of effect of systemic AMPK activation, we were interested in a nanoparticle-based targeting of direct AMPK activator MK-8722 (NP-MK8722) delivery. We determined that PLGA-PEG nanoparticles (NPs) accumulated in hepatic macrophages as early as 2 hours post-injection, but NP-MK8722 did not alter hepatic necrosis, injury, or immune infiltration. Overall, my thesis work has advanced our knowledge of the effects of housing temperatures on NASH pathogenesis. Moreover, we are the first to address the effects of macrophage AMPK signaling in NASH and AILI. This is especially true for assessing how AMPK deficiency and targeted activation influences KC immunometabolism during injury.

PhD Dissertation / Obesity is a major risk factor for type 2 diabetes (T2D) and non-alcoholic fatty liver disease (NAFLD), and is attributed to excess energy intake in comparison to energy expenditure. Therapeutics that reduce energy intake in obesity have limited efficacy, with weight loss typically reaching less than 10% of initial body mass, leading to efforts to uncover new therapies that may increase energy expenditure. Unlike lipid-storing white adipose tissue, brown and beige adipose tissues undergo futile cycling, oxidizing lipids and carbohydrates thereby increasing energy expenditure. With obesity, the metabolic activity of brown and beige adipose tissue is reduced, suggesting that restoring adipose tissue thermogenesis may represent a new means to enhance energy expenditure. Previous studies in mice have shown that peripheral serotonin synthesis by the enzyme tryptophan hydroxylase 1 (Tph1) inhibits adipose tissue thermogenesis and contributes to the development of obesity, insulin resistance and NAFLD. However, the primary Tph1 expressing tissue(s) inhibiting adipose tissue futile cycling is not known. In this thesis, we genetically removed Tph1 in mast cells of mice and discovered that this elevated beige adipose tissue activity protecting mice from developing high-fat diet induced obesity, insulin resistance and NAFLD. In contrast to these findings, genetic deletion of Tph1 in adipocytes did not result in protection from obesity, suggesting that mast cells are the primary source of serotonin that inhibits white adipose tissue thermogenesis. Lastly, to determine the importance of adipose tissue thermogenesis in mediating the beneficial metabolic effects of reduced Tph1, mice were housed at thermoneutrality, blocking the requirement for adipose tissue thermogenesis. Under these conditions, mice lacking Tph1 had comparable brown and beige adipose tissue metabolic activity, energy expenditure and adiposity, however, surprisingly, were still protected from insulin resistance and NAFLD. The studies in this dissertation have discovered that mast cell Tph1 is critical for inhibiting adipose tissue thermogenesis and that serotonin plays an important role in promoting NAFLD, independently of its inhibitory effects on adipose tissue thermogenesis. Collectively, these findings further define the roles of serotonin in regulating whole-body energy metabolism, providing critical clues and mechanistic insights for potential therapies to mitigate metabolic diseases. / Dissertation / Doctor of Philosophy (Medical Science) / Obesity, type 2 diabetes (T2D) and non-alcoholic fatty liver disease (NAFLD) can develop when caloric intake exceeds expenditure. In contrast to lipid-storing white fat, brown and beige fat burn calories. Serotonin is a hormone that reduces the burning of calories in fat, therefore finding ways to inhibit its effects on fat tissue without altering serotonin in the brain may lead to new therapies for obesity and other related diseases. In this thesis, we examined potential sources of serotonin that might inhibit the burning of calories in adipose tissue of mice. By reducing the synthesis of serotonin in a white blood cell called mast cells, but not fat cells, mice were protected from obesity, pre-diabetes and NAFLD due to increased activity of beige fat. Moreover, when we kept mice in a warm environment, thus reducing the need for mice to burn calories in brown and beige fat, this eliminated the effects of serotonin to promote obesity, but not pre-diabetes and NAFLD. These studies have identified how serotonin generated from mast cells inhibits the burning of calories in adipose tissue, a finding that may lead to new therapies for obesity, T2D and NAFLD.

京都大学 / 新制・課程博士 / 博士(医学) / 甲第23603号 / 医博第4790号 / 新制||医||1055(附属図書館) / 京都大学大学院医学研究科医学専攻 / (主査)教授 上野 英樹, 教授 金子 新, 教授 河本 宏 / 学位規則第4条第1項該当 / Doctor of Medical Science / Kyoto University / DFAM

Les travaux de l'équipe ont permis de montrer qu'IL4I1 est une L-amino acide oxydase sécrétée par les cellules d'origine myéloïde dégradant la phénylalanine en H2O2, NH3 et phénylpyruvate. Elle est fortement exprimée au sein des tumeurs humaines et facilite l'échappement tumoral dans un modèle de mélanome murin. Cette enzyme inhibe l'expression de la chaîne ζ du TCR ainsi que la prolifération des lymphocytes T effecteurs/mémoires via la production d'H2O2. IL4I1 appartient donc à une famille d'enzymes régulatrices des réponses immunitaires impliquées dans la défaillance de la réponse anti-tumorale.Au cours de ma thèse, j'ai montré qu'IL4I1 induit la différenciation des lymphocytes T CD4+ naïfs conventionnels en cellules CD25fortFoxP3+ chez l'Homme et la souris. Ces cellules exercent une action suppressive in vitro équivalente à celle de cellules régulatrices obtenues sans IL4I1 et leur phénotype est similaire. La promotion de la différenciation Treg par IL4I1 a pu être observée dans différentes conditions in vitro et s'avère particulièrement importante lorsque les cellules sont cultivées sans ajout d'IL2 et de TGFβ. Le mécanisme impliqué reposerait en partie sur la consommation de Phe par l'activité enzymatique qui serait responsable de l'inhibition de la voie mTORC1 observée.En conclusion, nous avons démontré un nouveau rôle d'IL4I1 sur les lymphocytes T. Ainsi, en inhibant la prolifération des lymphocytes T et en induisant la polarisation Treg, IL4I1 pourrait jouer un rôle important dans l'échappement tumoral. IL4I1 étant sécrétée et peu exprimée à l'état physiologique, elle pourrait être la cible de traitements adjuvants dans le cancer. / Our team has shown that IL4I1 is a secreted L-amino acid oxidase which degrades phenylalanine into H2O2, NH3 and phenylpyruvate.. This enzyme is produced by myeloid cells and expressed within human cancers. IL4I1 expression facilitates tumor growth in a mouse model. IL4I1 inhibits TCRζ chain expression and T lymphocyte proliferation via H2O2 production. Therefore IL4I1 belongs to a family of enzymes endowed with immune regulatory functions involved in the anti-tumor response failure.During my PhD, I showed that IL4I1 induces CD25highFoxP3+ cells differentiation from conventional naïve CD4+ T cells, both in humans and mice in vitro systems. These cells exert similar in vitro suppressive activity than those obtained without IL4I1 with a similar phenotype. Treg differentiation promotion by L4I1 is observed in various in vitro conditions and is particularly important when cells are cultured without addition of IL2 and TGFβ. The involved mechanism would partially depend on the phenylalanine consumption by the enzymatic activity which would be responsible for the mTORC1 pathway inhibition observed.In conclusion, we have demonstrated a new mechanism of IL4I1 action on T lymphocytes. Thus, by inhibiting T lymphocytes proliferation and by inducing Treg polarization, IL4I1 could play an important role in tumor escape. Since IL4I1 is secreted and weakly expressed under physiological conditions, it could be the target of adjuvant therapy in cancer.

IFNγ is an essential and pleiotropic activator of monocytes, but little is known about the effects IFNγ on cellular metabolism. Therefore, we sought to characterize and elucidate the mechanisms by which IFNγ reprograms monocyte metabolism to support its immunologic activities. First, we identified a critical role for IFNγ in the induction of immunoresponsive gene 1 (IRG1) and its product, itaconate. The immunometabolite, itaconate, has been reported to have antibacterial, anti-inflammatory and antioxidant activity. Irg1-/- mice, lacking itaconate, are highly susceptible and phenotypically similar to IFNγ knock out (GKO) mice upon infection with Mycobacterium tuberculosis. Therefore, we assessed the role of IRG1/itaconate in the context of non-tuberculous mycobacterial (NTM) infection, the most common type of infection in patients with immunodeficiencies caused by defects in IFNγ signaling. Our data suggest that impaired induction of itaconate in the context of mycobacterial infection may contribute to mycobacterial susceptibility and immune dysregulation in patients with defects in IFNγ signaling. Next, we evaluated the metabolic phenotype of IFNγ-stimulated human monocytes and found that IFNγ increased oxygen consumption rates (OCR), indicative of reactive oxygen species generation by both mitochondria and NADPH oxidase. Transcriptional profiling of human macrophages revealed that this oxidative phenotype was dependent on IFNγ-induced, nicotinamide phosphoribosyltransferase (NAMPT)-mediated NAD+ salvage to generate NADH and NADPH for oxidation by mitochondrial complex I and NADPH oxidase, respectively. These data identify an IFNγ-induced, NAMPT-dependent, NAD+ salvage pathway that is critical for complete induction of the respiratory burst in IFNγ stimulated human monocytes.

Despite our growing knowledge of virus biology they continue to present a problem to global public health. This problem arises from their high mutation rates that allow them to evade antiviral therapies that we have developed to date. An alternative solution for developing antiviral therapies could be to target host cell factors that are hijacked by the virus. The basis of this hypothesis is that if we can stop the virus from using host cell machinery or from evading host immune mechanisms we could treat the infection more efficiently. With the major research focus being on viral proteins and how we can prevent their functions, there is a lot of work to be done in finding host factors that could be the key to treating an infection. The three themes presented in this thesis broadly focus on this goal. The first theme looks at miRNAs, their interacting partners, and their dysregulation during HCV infection. A microRNA is identified from a small molecule screen of miRNAs that are dysregulated during HCV infection and its role in liver immunometabolism is examined to determine its antiviral potential and identify host factors that could be of interest to target with antiviral therapeutics. The second theme examines the potential of activity-based protein profiling techniques for complementing existing antiviral therapies. An azauracil probe is characterized to examine its ability to interact with viral polymerases and its suitability as a building block for antiviral research or therapies. The final theme uses activity-based protein profiling techniques to study a novel carbamate-hydrazone chemotype and establish its suitability as a chemical probe. The hydrazone probe’s reactivity with the mammalian proteome was determined and its interacting partners were identified using chemoproteomic techniques with an overall goal of examining its suitability for antiviral research. Overall, this thesis uses chemical and molecular biology techniques to present three differing perspectives on how to approach the discovery of host factors and develop novel antiviral therapies.