Abstract
Alternatively activated macrophages are innate immune cells that contribute to resolution of inflammation and maintenance of homeostasis. Modulation of available fatty acid sources is thought to affect cellular physiology through a variety of mechanisms, including through alterations to the profile of oxygenated free fatty acid metabolites, called oxylipins, produced in a cell type specific manner. Here, we investigated how treatment with the plant-sourced omega-3 fatty acid α-linolenic acid (ALA) affects the oxylipin profile and functional capacity of a cell culture model of human alternatively activated (M2a-like) macrophages. In a targeted but unbiased screen, ALA enhanced the production of oxylipins from all polyunsaturated fatty acid (PUFA) precursors, with oxylipins derived from ALA being enhanced the most. Consistently, ALA treatment enhanced the expression of both cytoplasmic and calcium-independent phospholipase A2. At a functional level, ALA treatment increased phagocytic activity and altered production of the chemokine MCP-1 by M2a-like cells in a manner dependent on the time of treatment. ALA treatment during polarization increased MCP-1 secretion, which was sensitive to pharmacological inhibition of 15-LOX-1 by ML351. Thus, ALA modulates the phenotype of alternatively activated macrophages, likely through its own LOX-derived oxylipins and/or through general modulation of oxylipin biosynthesis. These effects likely contribute to the overall anti-inflammatory benefit observed with ALA supplementation.
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Key Points
PUFAs are thought to exert their bioactive effects through multiple mechanisms, including directly via receptor mediated signaling, through structural alteration of cell membranes and by enzymatic and non-enzymatic conversion to oxygenated metabolites called oxylipins. Oxylipins then act through G protein coupled receptors or lipid binding transcription factors. EPA and DHA are substrates oxylipin derivatives that can antagonize the actions of AA derived oxylipins and/or initiate unique pro-resolving signaling pathways. Oxylipins derived from ALA have also been identified and may contribute to the overall bioactivities of ALA directly, without a need for conversion to longer chain EPA and DHA. In the current study, how ALA modulates oxylipin production by alternatively activated [M(IL4+IL13) referred to here as M2a-like] macrophages and how this relates to changes in cellular function were investigated. Treatment with ALA during polarization to the M2a-like state leads to a general increase in oxylipins from all precursor PUFAs, with ALA oxylipins and LOX products being the most markedly upregulated. Treatment of THP1-derived macrophages with ALA during polarization to the M2a-like state broadly increases oxylipins from all precursor PUFAs, especially those derived from ALA itself and those generated by the LOX enzyme family. Consistently, protein levels of cPLA2-α and iPLA2-β, enzymes that liberate free FAs from phospholipid, were also increased by ALA. These changes were accompanied by an increased ability to phagocytose bioparticles and by increased secretion of the chemokine MCP-1. The results show that several 15-LOX products are increased by ALA treatment, including, in order of decreasing magnitude, 13−HOTrE from ALA, 15-HEPE from EPA, 13−HODE from LA, 13−HOTrE-γ from DGLA and 15-oxo-eicosatetraenoic acid (oxo-ETE) from AA. The actions of omega-3 PUFA such as ALA on immune cells and surrounding tissues are complex and multi-faceted, but ultimately resolvable. This study presents an important advancement toward this goal by describing how the presence of ALA favourably alters the function of alternatively activated macrophages at least in part via changes to the profile of secreted oxylipins.