Biosynthèse de médiateurs lipidiques dérivés des lipoxygénases par les leucocytes : importance dans l'inflammation

Authors: Archambault, Anne-Sophie
Advisor: Flamand, NicolasLaviolette, Michel
Abstract: Lipid mediators play a central role in the regulation of inflammation. Some induce the migration and activation of leukocytes at the site of an infection or a danger, while others are involved in the resolution of inflammation, acting as anti-inflammatory and pro-resolving mediators. Many enzymes are responsible for the biosynthesis of these lipid mediators. Lipoxygenases (LO) metabolize fatty acids, endocannabinoids, and phospholipids into both pro- and anti-inflammatory bioactive lipid mediators, making LOs very interesting therapeutic targets for the treatment of chronic inflammation. In humans, there are many LOs, including 5-LO and 15-LO, both enzymes expressed in leukocytes. The main objective of the project was to characterize the production of LO-derived lipid mediators by leukocytes. Using LC-MS/MS, we quantified the production of LO-derived mediators by neutrophils and eosinophils. This project was divided in four sections with specific objectives. First, we characterized the production of 15-LO derived lipid mediators by human neutrophils and eosinophils. We demonstrated that both eosinophils and neutrophils can produce 15-LO-hydroxylated mediators from fatty acids (linoleic acid, dihomo-γ-linolenic acid, arachidonic acid, eicosapentaenoic acid and docosahexaenoic acid), and from endocannabinoids (2-arachidonoyl-glycerol and N-arachidonoyl-ethanolamine). Moreover, we characterized two new 15-LO-derived mediators, 13-HODE-G and 13-HODE-EA. We also showed that eosinophils produced 15-LO-hydroxylated metabolites through 15-LO-1, but the mechanism behind the production of these metabolites by neutrophils has not been defined. The second objective of the project was to identify the role of the 5-LO-derived mediator LTB₄, and the impact of its degradation. LTB₄ is a pro-inflammatory lipid mediator and is rapidly degraded into 20-OH- and 20-COOH-LTB₄ by CYP4F3A, an enzyme expressed in human neutrophils. We showed that LTB₄ metabolites are less effective than LTB₄ at inducing neutrophil functions. Moreover, these metabolites inhibited the LTB₄-dependent neutrophil and eosinophil functions. The metabolites induced the internalization of BLT₁, explaining the inhibition of the LTB₄-dependent functions. We also identified PF-4708671 as a new CYP4F3A inhibitor, that was more potent than the CYP4F3A commercial inhibitor 17-ODYA. PF-4708671 could therefore be used as a template to produce more specific CYP4F3A inhibitors. The third objective was to compare lipid mediator production between mouse and human eosinophils. Mouse models are widely used to investigate inflammatory diseases and the role of lipid mediators. We directly compared the biosynthetic ability of these cells to produce LO-derived lipid mediators. We found major differences in the production of these lipid mediators. Compared to human eosinophils, mouse eosinophils biosynthesized mainly LTB₄ (and almost no LTC₄), more 12-HETE (and less 15-HETE), and around 10 times more specialized pro-resolving mediators. Lastly, the fourth objective was to investigate the LO-derived lipid mediators in the context of acute inflammation. Because part of this project was done during the COVID-19 pandemic, we quantified the LO-derived mediators in the bronchoalveolar lavage fluids of severe COVID-19 patients and compared the levels with healthy donors. We found that all the LO-derived mediators and their substrates were increased in the lung of COVID-19 patients. Moreover, almost all the lipid mediators we quantified correlated with each other, showing that there is a lipid storm in the lungs of severe COVID-19 patients. In conclusion, this project increases the knowledge of LO-derived lipid mediator biosynthesis in neutrophils and eosinophils and on how to modulate their levels. Lipid mediators are complex and can have both pro- and anti-inflammatory effects. A better understanding of the biosynthesis of these lipids is highly important and could lead to the development of new anti-inflammatory therapies modulating their levels.
Document Type: Thèse de doctorat
Issue Date: 2022
Open Access Date: 9 May 2022
Grantor: Université Laval
Collection:Thèses et mémoires

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