A Phosphatidylinositol Species Acutely Generated by Activated Macrophages Regulates Innate Immune Responses

Abstract

Activation of macrophages with stimuli of the innate immune response results in the intense remodeling of arachidonate-containing phospholipids, leading to the mobilization of large quantities of this fatty acid for conversion into biologically active eicosanoids. As a consequence of this process, the arachidonate levels in membrane phospholipids markedly decrease. We have applied mass spectrometry–based lipid profiling to study the levels of arachidonate-containing phospholipids under inflammatory activation of macrophages. We identify an unusual inositol phospholipid molecule, PI(20:4/20:4), the levels of which do not decrease but actually increase by 300% after activation of the macrophages. PI(20:4/20:4) is formed and degraded rapidly, suggesting a role for this molecule in regulating cell signaling events. Using a metabolipidomic approach consisting in exposing the cells to deuterium-labeled arachidonate at the time they are exposed to stimuli, we show that PI(20:4/20:4) biosynthesis occurs via the sequential incorporation of arachidonate, first into the sn-2 position of a preformed phosphatidylinositol (PI) molecule, followed by the rapid introduction of a second arachidonate moiety into the sn-1 position. Generation requires the participation of cytosolic phospholipase A2α and CoA-dependent acyltransferases. PI(20:4/20:4) formation is also detected in vivo in murine peritonitis exudates. Elevating the intracellular concentration of PI(20:4/20:4) by introducing the lipid into the cells results in enhancement of the microbicidal capacity of macrophages, as measured by reactive oxygen metabolite production and lysozyme release. These findings suggest that PI(20:4/20:4) is a novel bioactive inositol phospholipid molecule that regulates innate immune responses in macrophages.

Group V Secreted Phospholipase A2 Is Upregulated by IL-4 in Human Macrophages and Mediates Phagocytosis via Hydrolysis of Ethanolamine Phospholipids

“…PE and PI molecular species were identified by multiple reaction monitoring experiments on chromatographic effluent by comparison with previously published data (24,(26)(27)(28)(29)(30)(52)(53)(54). Cutoff parameter was set at m/z 150, and fragmentation amplitude was set at one arbitrary unit.…”

“…In previous work, we investigated the mechanisms of PLA 2 -mediated phospholipid turnover in monocytes and macrophages responding to a variety of stimuli of innate immunity and inflammation (21)(22)(23)(24)(25)(26)(27)(28)(29)(30). In those studies we took advantage of mass spectrometry-based lipidomic approaches to define, at a molecular species level, the phospholipid substrate specificities of the enzymes involved.…”

Cytosolic Group IVA and Calcium-Independent Group VIA Phospholipase A2s Act on Distinct Phospholipid Pools in Zymosan-Stimulated Mouse Peritoneal Macrophages

“…Ethanolamine glycerophospholipids (PE) species and phosphatidylinositol (PI) were detected in negative ion mode with the capillary current set at +3500 V over the initial 25 min as [M2H] 2 ions. Choline glycerophospholipid (PC) species were detected over the elution interval from 25 to 35 min in positive ion mode, as [M+H] + ions, with the capillary current set at 23500 V. PE and PI molecular species were identified by multiple reaction monitoring experiments on chromatographic effluent by comparison with previously published data (32)(33)(34)(35)(36)(37)(38). Cutoff parameter was set at m/z 150, and fragmentation amplitude was set at 1 arbitrary unit.…”

“…For the identification of acyl chains of PC species, ionization was carried out in negative mode with postcolumn addition of acetic acid at a flow rate of 100 ml/h as [M+CH 3 CO 2 ] 2 adducts, and acyl chains were identified by MS 3 experiments. Stereospecific assignation of fatty acyl chains was carried out by comparing the relative intensities of the 1-lysophospholipid and 2-lysophospholipid compounds arising in the fragmentation experiments (the signal of the latter predominates over that of the former in ion-trap MS) (32)(33)(34)(35)(36)(37)(38).…”

“…In those studies (32)(33)(34)(35)(36)(37)(38), we have taken advantage of mass spectrometry (MS)-based lipidomic approaches to define, at a molecular species level, the sources of AA liberation and incorporation. In this study, we have applied a similar approach to relate changes in phospholipid turnover with the activation of particular PLA 2 forms: cPLA 2 a and iPLA 2 b.…”