Myeloid-Specific Deficiency of Long-Chain Acyl CoA Synthetase 4 Reduces Inflammation by Remodeling Phospholipids and Reducing Production of Arachidonic Acid-Derived Proinflammatory Lipid Mediators

Abstract

In response to infection or tissue damage, resident peritoneal macrophages (rpMACs) produce inflammatory lipid mediators from the polyunsaturated fatty acid (PUFA), arachidonic acid (AA). Long-chain acyl-CoA synthetase 4 (ACSL4) catalyzes the covalent addition of a CoA moiety to fatty acids, with a strong preference for AA and other PUFAs containing three or more double bonds. PUFA-CoA can be incorporated into phospholipids, which is the source of PUFA for lipid mediator synthesis. In this study, we demonstrated that deficiency of Acsl4 in mouse rpMACs resulted in a significant reduction of AA incorporated into all phospholipid classes and a reciprocal increase in incorporation of oleic acid and linoleic acid. After stimulation with opsonized zymosan (opZym), a diverse array of AA-derived lipid mediators, including leukotrienes, PGs, hydroxyeicosatetraenoic acids, and lipoxins, were produced and were significantly reduced in Acsl4-deficient rpMACs. The Acsl4-deficient rpMACs stimulated with opZym also demonstrated an acute reduction in mRNA expression of the inflammatory cytokines, Il6, Ccl2, Nos2, and Ccl5 When Acsl4-deficient rpMACs were incubated in vitro with the TLR4 agonist, LPS, the levels of leukotriene B₄ and PGE₂ were also significantly decreased. In LPS-induced peritonitis, mice with myeloid-specific Acsl4 deficiency had a significant reduction in leukotriene B₄ and PGE₂ levels in peritoneal exudates, which was coupled with reduced infiltration of neutrophils in the peritoneal cavity as compared with wild-type mice. Our data demonstrate that chronic deficiency of Acsl4 in rpMACs reduces the incorporation of AA into phospholipids, which reduces lipid mediator synthesis and inflammation.

Figure 1.

Myeloid-specific knockout of Acsl4 reduces ACSL activity toward AA and alters AA content during homeostatic lipid remodeling in rpMACs. (A) mRNA expression of Acsl1–5. (B) Total membrane ACSL activity with 1–14C-AA. Protein pooled from 3 mice and assay performed in triplicate. (C-E) Incubation of rpMacs with 1–14C-AA for 18 hrs (pulse), washed with 2% BSA in PBS, then incubated for an additional 6 hours in 0.5% BSA in DMEM (chase). (C) Radiolabel released into media after a 6 hr chase, and (D) radiolabel retained in cellular lysate. (E) Free arachidonic acid measured by LC MS/MS in rpMACS. (F) Expression of fatty acid desaturase 1 and 2 (Fads1 & 2) and elongation of very long-chain fatty acid protein 5 (Elovl5) in rpMACs. Results are expressed as mean ± SEM; n = 4 unless otherwise stated. *p < 0.05, **p < 0.01, and ***p < 0.001 by Student’s t-test.

Figure 2.

Loss of Acsl4 reduces 20:4, 22:5, 22:6 fatty acids from phospholipids in rpMACs. Phospholipids were extracted from isolated rpMACs and analyzed by LC-MS/MS. Fatty-acyl species of (A) phosphatidylethanolamine (PE), (B) Phosphatidylcholine (PC), or (C) Phosphatidic acid (PA). D = diacyl phospholipids, P = plasmalogens (alkenyl acyl phospholipids), A = alkyl acyl phospholipids. Results are expressed as mean ± SEM; n = 4, *p < 0.05, **p < 0.01, and ***p < 0.001 by Student’s t-test. * p ≤ 0.05, ** p ≤ 0.01, *** p ≤ 0.001 by student’s t-test.

Figure 3.

Loss of Acsl4 reduces AA-derived lipid mediator production in rpMACs. rpMacs were isolated and treated with opsonized zymosan (opZym) (10 particles/cell) for 2 hours, reaction was quenched with 2 volumes cold methanol and lipid mediators were measured by LC-MS/MS. Results are expressed as mean ± SEM; n = 3–4, *p < 0.05, **p < 0.01, and ***p < 0.001 ****p < 0.0001 by two-way ANOVA with Tukey’s multiple comparisons. Arachidonic acid (AA), prostaglandin (PG), thromboxane (TX), lipoxin (LX), leukotriene (LT), hydroxyeicosatetraenoic acid (HETE), cyclooxygenase (COX), acetylated (ac), lipoxygenase (LOX), LTA4 hydrolase (LTA4H), non-enzymatic hydrolysis (non-enz. hydrolysis).

Figure 4.

Loss of Acsl4 blunts inflammatory gene expression in rpMACs. (A) Gene expression of key enzymes in the synthesis of LTB4 (5-lipoxegenase activating protein, Alox5ap; 5-lipoxegenase, Alox5; LTA4 hydrolase, Lta4h; prostaglandin-endoperoxide synthase, Ptgs2). The mRNA was normalized to GAPDH. (B) Gene expression of inflammatory markers throughout the time course of opsonized zymosan (10 particles/cell) stimulation at 0, 6, and 24 hrs. Results are expressed as mean ± SEM; n = 3–4. *p < 0.05, **p < 0.01, and ***p < 0.001 ****p < 0.0001 by Student’s t-test or repeated measures two-way ANOVA with Sidak’s multiple comparisons.

Figure 5.

LPS-induced neutrophil infiltration is reduced in the peritoneum with the loss of Acsl4. (A) rpMACs were isolated and treated with LPS (1µg/mL) for 6 hrs and medium was assayed by ELISA. Two-way ANOVA with Tukey’s multiple comparisons, *p ≤ 0.05, n = 3–4/group. (B) Mice were i.p. injected with LPS (10 mg/kg) and peritoneal lavage fluid was assayed for LTB4 and PGE2 after 4 hrs. (C) FACS analysis of peritoneal neutrophils (PMN) in the mice injected with LPS. Results are expressed as mean ± SEM; n = 3–4, *p < 0.05, by Student’s t-test unless otherwise noted. MACs, macrophages; PMN, polymorphonuclear cells.