Phospholipase A2 regulates eicosanoid class switching during inflammasome activation Paul C. Norrisa, David Gosselinb, Donna Reichartb, Christopher K. Glassb, and Edward A. Dennisa,1 Departments of aChemistry/Biochemistry and Pharmacology, and bCellular and Molecular Medicine, University of California, San Diego, La Jolla, CA 92093 Edited by Michael A. Marletta, The Scripps Research Institute, La Jolla, CA, and approved July 30, 2014 (received for review March 13, 2014) Initiation and resolution of inflammation are considered to be to initiate pathogenic killing, subsequent “class switching” to lipoxin tightly connected processes. Lipoxins (LX) are proresolution lipid (LX) formation by “reprogrammed” neutrophils inhibits additional mediators that inhibit phlogistic neutrophil recruitment and pro- neutrophil recruitment during self-resolving inflammatory resolu- mote wound-healing macrophage recruitment in humans via tion (9). The direct link between inflammatory commitment and potent and specific signaling through the LXA4 receptor (ALX). resolution mediated by eicosanoid signaling in macrophages One model of lipoxin biosynthesis involves sequential metabolism remains unclear from short-term vs. long-term priming, but the of arachidonic acid by two cell types expressing a combined trans- complete temporal changes and important interconnections cellular metabolon. It is currently unclear how lipoxins are effi- within the entire eicosadome are now demonstrated. ciently formed from precursors or if they are directly generated after receptor-mediated inflammatory commitment. Here, we pro- Results vide evidence for a pathway by which lipoxins are generated in We first primed immortalized macrophage-like cells (RAW264.7) macrophages as a consequence of sequential activation of toll-like with the TLR4 agonist Kdo2 lipid A (KLA) for various times and receptor 4 (TLR4), a receptor for endotoxin, and P2X7, a purinergic examined the effects on subsequent purinergic stimulated COX receptor for extracellular ATP. Initial activation of TLR4 results in and 5-LOX activity using targeted lipidomic monitoring (Fig. 1A). – accumulation of the cyclooxygenase-2 derived lipoxin precursor Total 5-LOX products (5-HETE, LTC4, 11-trans LTC4,LTB4, 15-hydroxyeicosatetraenoic acid (15-HETE) in esterified form 6-trans,12-epi LTB4, 6-trans LTB4, and 12-epi LTB4) peaked at within membrane phospholipids, which can be enhanced by aspi- 2 h and diminished steadily at later time points; total levels from BIOCHEMISTRY rin (ASA) treatment. Subsequent activation of P2X7 results in effi- 12 to 24 h were less than 1% of maximal 2-h levels. Total COX cient hydrolysis of 15-HETE from membrane phospholipids by products [PGD2, PGE2, PGF2α, PGJ2, 15-deoxy PGD2, 15-deoxy group IVA cytosolic phospholipase A , and its conversion to bio- 2 PGJ2, 11-hydroxyeicosatetraenoic acid (11-HETE) and 15- active lipoxins by 5-lipoxygenase. Our results demonstrate how HETE] were lowest during short-term TLR4 priming and a single immune cell can store a proresolving lipid precursor and then steadily increased with longer priming durations. AA levels in release it for bioactive maturation and secretion, conceptually similar media were maximal with 2-h priming and were vastly reduced to the production and inflammasome-dependent maturation of the with 8-h priming and beyond (Fig. S1); AA release during major proinflammatory IL-1 family cytokines. These findings provide evi- COX activity from 2 to 12 h may therefore be slower and/or dence for receptor-specific and combinatorial control of pro- and coupled to COX-2. anti-inflammatory eicosanoid biosynthesis, and potential avenues The proresolution mediators lipoxin A4 (LXA4) and 15-epi– to modulate inflammatory indices without inhibiting downstream LXA4 were also detected between 4 and 10 h of TLR4 priming eicosanoid pathways. and peaked at 8 h (Fig. 1A). LXA4 and 15-epi–LXA4 are trihy- droxylated eicosanoids derived from 15(S)-HETE and 15(R)- lipidomics | enzyme coupling | membrane remodeling HETE, respectively. The 15-HETE comprises ∼1–3% COX-2 complex network of danger-sensing receptors and bioactive Significance Apeptide and lipid signals, including cytokines and eicosa- noids, regulates innate immunity. Toll-like receptor (TLR) Group IVA cytosolic phospholipase A2 (GIVA cPLA2) is widely priming is suggested as a precautionary step in building a signif- viewed as the primary enzyme responsible for inflammatory icant inflammatory response by driving production of IL-1 family arachidonic acid (AA) release and with high specificity. Our protokines, which remain inactive until a second stimulus drives results demonstrate dual, phase-specific release of AA and them to bioactive maturation and secretion (1). The second step 15-hydroxyeicosatetraenoic (15-HETE) acid by GIVA cPLA2 in of this process has been most strongly linked to extracellular primary and immortalized macrophages during a receptor- ATP and specifically to one of its purinergic receptors, P2X7 (2, mediated program required for complete inflammatory com- 3), particularly in macrophages (4). mitment. These dual actions by GIVA cPLA2 were necessary for TLR stimulations also increase prostaglandin synthesis 2+ biosynthesis of proresolving lipoxins, providing a unique, up- by activating cytosolic phospholipase A2 (cPLA2) through a Ca - stream example of an enzyme linked to both the initiation and independent mechanism to release arachidonic acid (AA) from resolution of inflammation. Further, our results demonstrate phospholipids, and by increasing expression of cyclooxygenase-2 a single-cell mechanism of lipoxin synthesis that is more effi- (COX-2) and microsomal prostaglandin E2 synthase-1. P2X7 stim- cient than the established transcellular biosynthetic mechanisms, 2+ ulation activates cPLA2 through a Ca -dependent mechanism that underscoring the importance of enzyme coupling and the possi- couples AA metabolism with 5-lipoxygenase (5-LOX)-activating + bility of proresolution therapies at the membrane level. protein (FLAP), Ca2 -activated 5-LOX, and constitutive COX-1 to form leukotrienes (LTs) and prostaglandins (PGs). Short-term Author contributions: P.C.N. and E.A.D. designed research; P.C.N., D.G., and D.R. performed 2+ (∼1 h) TLR priming of Ca ionophore/P2X7-activated immune research; P.C.N. and D.R. analyzed data; and P.C.N., C.K.G., and E.A.D. wrote the paper. cells enhances LT synthesis (5, 6), but long-term TLR priming The authors declare no conflict of interest. (16–18 h) significantly suppresses LT synthesis by different cell- This article is a PNAS Direct Submission. type–specific mechanisms (7, 8). 1To whom correspondence should be addressed. Email: [email protected]. Whereas PGE2, PGI2, and LTC4 promote local edema from This article contains supporting information online at www.pnas.org/lookup/suppl/doi:10. postcapillary venules, and LTB4 amplifies neutrophil recruitment 1073/pnas.1404372111/-/DCSupplemental. www.pnas.org/cgi/doi/10.1073/pnas.1404372111 PNAS Early Edition | 1of6 Downloaded by guest on September 30, 2021 A levels of COX-2 after TLR4 stimulation (10). RPMs produced lipoxins with long-term priming (Fig. S4 A and B), and increasing 6000 KLA ATP Quench 0.12 Lipoxins cell density increased the level (and concentration) of PGE2, but this did not limit lipoxin formation or 5-LOX metabolism of AA 4000 Time 0.08 based on levels of LTC4 (Fig. S4 C and D). Thus, RPM and cells) 6 RAW macrophages both retain 5-LOX activity in the presence Total COX of exogenous or endogenous PGE , unlike in dendritic cells (8). 2000 Products 0.04 2 Eicosanoid Ultimately, lipoxins from macrophages likely represent an addi- (pmol/10 TotalTotal 5-LOX5-LOX ProductsProducts tional source of the total that might be found in certain physio- 0 0 logical environments. Lipoxins can be formed by coordinate 02481216 20 402481216 20 4conversion of endothelial COX-2/mucosal epithelial 15-LOX– Duration of TLR4 Priming (Hr) derived 15-HETE with neutrophil 5-LOX, or neutrophil 5-LOX– Total 5-LOX derived LTA with platelet 12-LOX, which inhibit neutrophil B PGD Lipoxins 4 2 Products extravasation (12). Macrophages initially recruit neutrophils via 500 0.25 100 Control leukotriene and chemokine signaling in response to TLR signaling 400 0.20 75 Celecoxib and may subsequently switch to forming lipoxins to inhibit neu- cells) 6 300 0.15 *** trophil recruitment in response to high ATP levels. ** 50 200 *** 0.10 To assess the enzymatic control of lipoxin formation, chiral Eicosanoid chromatography was used to determine the proportions of 15(R)- 100 0.05 25 (pmol/10 * N.D. HETE and 15(S)-HETE in TLR4 primed/purinergic-stimulated 0 0 0 1 7.5 1 7.5 1 7.5 RAW cells in the presence and absence of aspirin (ASA). Acet- ylation of COX-2 by ASA inhibits PG formation and enhances Duration of TLR4 Priming (Hr) 15(R)-HETE formation (13). Non–ASA-treated cells produced Fig. 1. Duration of TLR4 priming controls purinergic 5-LOX product for- 15(R)- and 15(S)-HETE at a ratio between 1:3 and 1:4 (R:S) mation and lipoxin biosynthesis. (A, Inset) Protocol for TLR4 priming (Kdo2 (Fig. 2A),andproducedbothlipoxinepimersataratioof∼1:2 lipid A, KLA) starting at time = 0 followed by ATP stimulation at indicated (15-epi–LXA4:LXA4). In the presence of ASA, RAW cells pro- times and subsequent reaction quench as endpoint (further details can be duced almost exclusively 15(R)-HETE and 15-epi–LXA4. These found in SI Materials and Methods); eicosanoid levels from RAW264.7 (RAW) results demonstrate that COX-2 activity with or without aspirin cell medium after TLR4 priming with 100 ng/mL KLA for varying durations – before stimulation with 2 mM ATP for the final 10 min include total COX treatment can lead to formation of 15-epi LXA4, which is more products (PGD2, PGE2, PGF2α, PGJ2, 15-deoxy PGD2, 15-deoxy PGJ2, 11-HETE, slowly inactivated by 15-hydroxyprostaglandin dehydrogenase and 15-HETE); total 5-LOX products (5-HETE, LTC4, 11-trans LTC4,LTB4, (PGDH) than LXA4 (12). Lipoxins are well known to be formed – 6-trans,12-epi LTB4, 6-trans LTB4, and 12-epi LTB4); lipoxins (LXA4 and 15-epi by either 12- or 15-LOX activity along with 5-LOX, although – LXA4).