This proposal focuses on the physiologic condition of sepsis, which is the tenth leading cause of death in the US with a mortality rate of >215,000 patients a year. For this reason, numerous mono-centric clinical trials (i.e. one therapeutic) have been undertaken, but these trials have shown limited success. As such, a critical need still exists for new therapeutic approaches and treatments for sepsis. Elucidation of new molecular mechanisms controlling inflammation provides the necessary foundation upon which to build. In this regard, an early manifestation of sepsis is the development of an imbalance between pro- and anti-inflammatory lipids such as the arachidonic acid (AA)-derived eicosanoids and 3-polyunsaturated fatty acid (PUFA)-derived lipid mediators (e.g. eicosapentaenoic acid (EPA)-derived (e.g. E-resolvins) and docosahexaenoic acid (DHA)- derived (e.g. D-resolvins)). The synthesis of these lipid mediators begins with the initial rate-limiting step, the formation of AA, EPA, and DHA via the activity of a phospholipase A2 (PLA2). One of the major PLA2s involved in this initial step is group IVA cytosolic PLA2 (cPLA2? and the Chalfant laboratory demonstrated that the sphingolipid, ceramide-1-phosphate (C1P), directly binds and activates cPLA2? the cationic ? groove of the C2 domain. To evaluate the physiological relevance of this lipid:protein interaction, we created a knockin mouse with the endogenous C1P interaction site of cPLA2ablated (KI). Intriguingly, our preliminary data showed that KI mice, unlike the wild-type (WT) and cPLA2? knockout (KO) mice, exhibit complete resistance to sepsis including marked reduction in neutrophil infiltration into the peritoneum. Notably, these mice displayed lower levels of pro-inflammatory eicosanoids with concomitant increases in anti-inflammatory resolvins. This novel lipid profile, in contrast to WT and KO mice, correlates strongly with lipid profiles of human patients that recover from sepsis. Mechanistically, the KI mouse also demonstrated differential usage in the phospholipid precursors utilized for DHA and EPA generation suggesting a previously unknown/unsurmised modulating role for C1P in cPLA2? function/localization. Based on our preliminary findings, we hypothesize that the sepsis resistance of KI mice and associated differential synthesis of specific pro- and anti-inflammatory lipid mediators: i) will b reflected by a """"""""perfect storm"""""""" of lipid mediator production in human patients who recover from sepsis, which preserves endothelium function by suppressing neutrophil trans-endothelial migration;thereby limiting the hyper-inflammatory stage of sepsis;ii) will reflect a novel """"""""lipid class switch"""""""" in the use of phospholipid substrates involving differential localization of regulatory and lipid synthetic proteins;and iii) will show specific and direct interaction of the C1P headgroup and sphingoid with cPLA2? upon co-crystallization experiments, which will allow for the future rational design of new therapeutics. We will validate these hypotheses and inferences using a multi-disciplinary approach including cellular, biophysical, structural, biological, and pr-clinical experiments.
We are focusing on the physiologic condition of sepsis, which is a term used to describe a severe illness arising from serious infection. The mortality rate of sepsis is >215,000 patients a year, and unfortunately, clinical trials for treating sepsis have shown limited success. As such, there is a major need for new therapeutics, and our studies explore the cellular mechanisms and bioactive lipids involved in ameliorating this deadly condition.
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