Differential contribution of lipoxygenase isoforms to the sequential biosynthesis of specialized pro-resolving...

Oxylipins, produced from polyunsaturated fatty acids (PUFA) by lipoxygenases (LOX), play pivotal roles in inflammation. While 5-LOX-derived leukotrienes promote inflammation, the specialized pro-resolving mediators (SPMs) contribute to inflammation resolution. The biosynthesis of the SPM members lipoxins and resolvins (RVs) supposedly relies on two sequential PUFA oxygenation steps, involving metabolism by 5-LOX and 12-/15-lipoxygenating paralogues (e.g., 12-LOX, 15-LOX-1/-2). These oxygenations may occur within one cell where both LOX isoforms are expressed or via transcellular metabolism between different cell types with complementary LOX expression. Here, we investigated monocellular incubations and (patho-)physiologically relevant co-incubations of human 5-LOX-positive polymorphonuclear leukocytes (PMNL) or monocytes with 12-LOX-positive platelets or with 15-LOX-1-rich M2a or dexamethasone-treated 15-LOX-2-rich M_Dex macrophages, reflecting possible transcellular SPM-biosynthetic routes. Comprehensive metabololipidomics using UPLC-MS/MS was employed for oxylipin profiling, and LOX-transfected HEK293 cells served as supportive model. While we confirmed the interplay of 5-LOX and 12-LOX in lipoxin formation in PMNL-platelet and monocyte-platelet co-incubations, we uncovered cooperation between 5-LOX and 15-LOX-2 yielding strong RvD5 and RvE4 formation in PMNL/M_Dex co-incubations. In contrast, 15-LOX-1-rich M2a produced substantial RvD5, while co-incubations with PMNL surprisingly impaired formation of RvD5 and other 15-LOX products. Moreover, we show that 15-LOX-1 prefers arachidonic acid and eicosapentaenoic acid as substrates, while 15-LOX-2 favors docosahexaenoic acid and 5- or 7-monohydroxylated precursors for SPM formation; in this context, 5- or 7-lipoxygenation is seemingly the first and rate-limiting oxygenation step. Conclusively, 15-LOX-1 and -2 isoforms exert distinct roles in conjunction with 5-LOX in the sequential biosynthesis of SPMs, which differs in monocellular versus co-incubations.