Polarization of Macrophages toward M2 Phenotype Is Favored by Reduction in iPLA2β (Group VIA Phospholipase A2)

Jason W Ashley,William D Hancock,Alexander J Nelson,Robert N Bone,Hubert M Tse,Mary Wohltmann,John Turk,Sasanka Ramanadham

doi:10.1074/jbc.m116.754945

Abstract

Macrophages are important in innate and adaptive immunity. Macrophage participation in inflammation or tissue repair is directed by various extracellular signals and mediated by multiple intracellular pathways. Activation of group VIA phospholipase A2 (iPLA2β) causes accumulation of arachidonic acid, lysophospholipids, and eicosanoids that can promote inflammation and pathologic states. We examined the role of iPLA2β in peritoneal macrophage immune function by comparing wild type (WT) and iPLA2β-/- mouse macrophages. Compared with WT, iPLA2β-/- macrophages exhibited reduced proinflammatory M1 markers when classically activated. In contrast, anti-inflammatory M2 markers were elevated under naïve conditions and induced to higher levels by alternative activation in iPLA2β-/- macrophages compared with WT. Induction of eicosanoid (12-lipoxygenase (12-LO) and cyclooxygenase 2 (COX2))- and reactive oxygen species (NADPH oxidase 4 (NOX4))-generating enzymes by classical activation pathways was also blunted in iPLA2β-/- macrophages compared with WT. The effects of inhibitors of iPLA2β, COX2, or 12-LO to reduce M1 polarization were greater than those to enhance M2 polarization. Certain lipids (lysophosphatidylcholine, lysophosphatidic acid, and prostaglandin E2) recapitulated M1 phenotype in iPLA2β-/- macrophages, but none tested promoted M2 phenotype. These findings suggest that (a) lipids generated by iPLA2β and subsequently oxidized by cyclooxygenase and 12-LO favor macrophage inflammatory M1 polarization, and (b) the absence of iPLA2β promotes macrophage M2 polarization. Reducing macrophage iPLA2β activity and thereby attenuating macrophage M1 polarization might cause a shift from an inflammatory to a recovery/repair milieu.

Highlights

Macrophage participation in inflammation or tissue repair is directed by various extracellular signals and mediated by multiple intracellular pathways
The family of PLA2s has been implicated in inflammatory responses and contribution to onset and/or progression of autoimmune-mediated disease [48, 49], and iPLA2␤ has recently been linked to diabetes [29]
Among its proposed roles in macrophages, iPLA2␤ has been implicated in playing a major role in free fatty acid accumulation in macrophages (60 – 63) leading to apoptosis. iPLA2␤, but not cPLA2, has been reported to promote macrophage proliferation [37]

Summary

Results

PLA2 Expression in Macrophages—PLA2s are ubiquitously expressed and activated in inflammatory settings [17, 37, 38], and as expected, iPLA2␤ and cPLA2 mRNA species increase during macrophage differentiation from their bone marrowderived precursors (Fig. 1A). Effects of iPLA2␤, COX, and 12-LO Inhibitors on M2 Markers—To assess the impact of eicosanoid-generating enzymes on eliciting an M2 phenotype, the effects of S-BEL, indomethacin, and CDC on select M2 markers (Arg, MRC1, STAT6, CCL2) in the absence and presence of alternative activation were examined in WT peritoneal macrophages. We find that under naïve conditions, NOX4 is reduced by 50% in iPLA2␤Ϫ/Ϫ relative to WT peritoneal macrophages (Fig. 11A), even in the presence of classical activation with IFN␥ ϩ LPS, NOX4 was 70% lower in iPLA2␤Ϫ/Ϫ, relative to WT macrophages These findings, taken together with nitrite accumulation presented, C and D) suggest that downstream generators of proinflammatory ROS are subject to modulation by iPLA2␤. These findings suggest an additional component in the inflammatory process, wherein feedback regulation of iPLA2␤ involves ROS

Discussion

Experimental Procedures

Mass Spectrometric Determination of LPA Molecular