UC San Diego

Oxidized polyunsaturated fatty acids represent an ever- growing class of lipid molecules comprised of hundreds, and likely thousands, of unique structural identities. Eicosanoids are the most well characterized oxidized lipids, which are derived from a 20-carbon, 4-double bond- containing fatty acid, arachidonic acid. Several eicosanoids are essential bioactive regulators of various physiological processes that promote advantageous human health outcomes, with inflammation being the most prominent. Pain, swelling, fever, and a host of other adverse conditions naturally accompany inflammation and are partly due to the actions of two well studied classes of eicosanoids, prostaglandins and leukotrienes. For obvious reasons, prostaglandins and leukotrienes have been among the most intensely pursued drug targets for several decades, but existing therapies have yielded mixed results, often at the cost of problematic, or even detrimental side effects. How eicosanoids and other oxidized lipids are biologically produced in animal and human inflammatory contexts is currently understood mostly at the in vitro level, which describes a general pathway of enzymes and receptors. Some eicosanoid pathway enzymes produce multiple lipid species, and some enzyme isomers only produce a single lipid species. Presently, a biologically relevant cellular understanding of eicosanoid production is not fully well developed, but the central importance of the biological roles of eicosanoids and related compounds in health and disease demands continued progress in this pursuit. Macrophage cells play a pivotal role in orchestrating innate immunity, partly through their ability to produce eicosanoids in response to both pathogenic and sterile traumatic cues. These haematopoietic immune cells proficiently sense danger signals via pathogen- recognizing receptors, and rid the body of excessive cellular accumulation via phagocytosis of apoptotic leukocytes during inflammation and red blood cells during homeostasis. In the case of eicosanoid production, cytokine production, and other functions, macrophages display remarkable phenotypic variability depending on their site of physiological residence. The focus of this thesis is three-fold. It serves to elucidate the factors that dictate inflammatory eicosanoid production at the cellular level for therapeutic gain, to explore the macrophage as a model cell for inflammatory lipid metabolism, and to seek a deeper meaning for the macrophage's inherent necessity. Central to these aims are two recently improved and expanded lipidomic analytical methods for eicosanoids and fatty acids that were employed and coupled with traditional molecular biology techniques. Differences in macrophage phenotypes were "lipidomically fingerprinted" using inflammatory receptor stimulation, and were also exploited to establish how prostaglandin and thromboxane synthesis varies based on enzyme transcript proportionality. Supplementation of different poly- unsaturated fatty acids was used to characterize the impact of substrate variability on eicosanoid signaling, and to more accurately describe the effects of omega-3 fish oil fatty acids in inflammation using two different inflammatory stimuli. Using these insights, a temporal and combinatorial receptor-mediated dependence for pro- resolution lipoxin synthesis was revealed, providing a likely role for macrophages in the continuum that drives the resolution of inflammation