SP24 Stressed lipids

SP24 Signalling and modulation of cell stress-induced lipid mediator biosynthesis

Project leader: Oliver Werz

Background and previous work
Innate immune cells possess high capacities for biosynthesis of bioactive lipid mediators involving the enzyme 5-lipoxygenase (5-LO) with either pro-inflammatory (e.g. leukotrienes) or anti-inflammatory/pro-resolving (e.g. lipoxins, resolvins) properties. We showed that cell stress activates 5-LO for leukotriene formation, involving MAPK signalling and 5-LO phosphorylation. While these pro-inflammatory leukotrienes are produced by 5-LO at the onset of stress response and cause damage, the anti-inflammatory lipoxins and resolvins are formed by the same enzyme at later time points, actively resolve inflammation, and may cause resistance and persistence. Current studies with human leukocytes and mouse peritonitis as disease model address how dose and duration of cell stress influences the spectrum of pro- and anti-inflammatory lipid mediator synthesis.

Specific aims and working programme
Based on recent data we hypothesize that nuclear 5-LO in a phosphorylated form generates pro-inflammatory leukotrienes in the early stress phase, whereas cytosolic 5-LO may generate pro-resolving lipoxins and resolvins in the later resolution phase. We speculate that the dose and duration of cell stress determines 5-LO subcellular localization (cytosolic/ nuclear) in leukocytes due to differential MAPK signalling and distinct interaction with specific 5-LO-binding proteins. We will study lipid mediator synthesis in primary human neutrophils, monocytes, macrophages, and B cells. Stress stimuli include genotoxic agents (e.g., arsenite), inflammatory cytokines, ROS and osmotic shock at short (0.5 to 10 min), mid (0.5 - 5 hours), and long term (0.5 - 6 days). UPLC-MS/MS [58] will be used to analyse lipid mediator species. 5-LO subcellular localization and interaction with specific 5-LO-binding proteins will be studied by immunofluorescence microscopy and proximity ligation assay, respectively. Mouse peritonitis induced by inflammatory stress (dose- and time-dependent) will serve as disease model for in vivo proof-of-concept studies. Exudates will be assessed for lipid mediators by UPLC-MS/MS. Because testosterone limits 5-LO activity in neutrophils and monocytes involving ERK-1/2, we hypothesize that testosterone may modulate stress responses by affecting lipid mediator synthesis with consequent sex differences. The molecular mechanism of testosterone-induced ERK-1/2 activation and thus subcellular redistribution of 5-LO shall be revealed. Recent own data suggest that testosterone may facilitate the interaction between tyrosine kinases upstream of Ras.