Phospholipase A2 regulates eicosanoid class switching during inflammasome activation

Initiation and resolution of inflammation are considered to be tightly connected processes. Lipoxins (LX) are proresolution lipid mediators that inhibit phlogistic neutrophil recruitment and promote wound-healing macrophage recruitment in humans via potent and specific signaling through the LXA₄ receptor (ALX). One model of lipoxin biosynthesis involves sequential metabolism of arachidonic acid by two cell types expressing a combined transcellular metabolon. It is currently unclear how lipoxins are efficiently formed from precursors or if they are directly generated after receptor-mediated inflammatory commitment. Here, we provide evidence for a pathway by which lipoxins are generated in macrophages as a consequence of sequential activation of toll-like receptor 4 (TLR4), a receptor for endotoxin, and P2X₇, a purinergic receptor for extracellular ATP. Initial activation of TLR4 results in accumulation of the cyclooxygenase-2–derived lipoxin precursor 15-hydroxyeicosatetraenoic acid (15-HETE) in esterified form within membrane phospholipids, which can be enhanced by aspirin (ASA) treatment. Subsequent activation of P2X₇ results in efficient hydrolysis of 15-HETE from membrane phospholipids by group IVA cytosolic phospholipase A₂, and its conversion to bioactive lipoxins by 5-lipoxygenase. Our results demonstrate how a single immune cell can store a proresolving lipid precursor and then release it for bioactive maturation and secretion, conceptually similar to the production and inflammasome-dependent maturation of the proinflammatory IL-1 family cytokines. These findings provide evidence for receptor-specific and combinatorial control of pro- and anti-inflammatory eicosanoid biosynthesis, and potential avenues to modulate inflammatory indices without inhibiting downstream eicosanoid pathways.A complex network of danger-sensing receptors and bioactive peptide and lipid signals, including cytokines and eicosanoids, regulates innate immunity. Toll-like receptor (TLR) priming is suggested as a precautionary step in building a significant inflammatory response by driving production of IL-1 family protokines, which remain inactive until a second stimulus drives them to bioactive maturation and secretion (1). The second step of this process has been most strongly linked to extracellular ATP and specifically to one of its purinergic receptors, P2X₇ (2, 3), particularly in macrophages (4).TLR stimulations also increase prostaglandin synthesis by activating cytosolic phospholipase A₂ (cPLA₂) through a Ca²⁺-independent mechanism to release arachidonic acid (AA) from phospholipids, and by increasing expression of cyclooxygenase-2 (COX-2) and microsomal prostaglandin E₂ synthase-1. P2X₇ stimulation activates cPLA₂ through a Ca²⁺-dependent mechanism that couples AA metabolism with 5-lipoxygenase (5-LOX)-activating protein (FLAP), Ca²⁺-activated 5-LOX, and constitutive COX-1 to form leukotrienes (LTs) and prostaglandins (PGs). Short-term (∼1 h) TLR priming of Ca²⁺ ionophore/P2X₇-activated immune cells enhances LT synthesis (5, 6), but long-term TLR priming (16–18 h) significantly suppresses LT synthesis by different cell-type–specific mechanisms (7, 8).Whereas PGE₂, PGI₂, and LTC₄ promote local edema from postcapillary venules, and LTB₄ amplifies neutrophil recruitment to initiate pathogenic killing, subsequent “class switching” to lipoxin (LX) formation by “reprogrammed” neutrophils inhibits additional neutrophil recruitment during self-resolving inflammatory resolution (9). The direct link between inflammatory commitment and resolution mediated by eicosanoid signaling in macrophages remains unclear from short-term vs. long-term priming, but the complete temporal changes and important interconnections within the entire eicosadome are now demonstrated.