Dormant 5-lipoxygenase in inflammatory macrophages is triggered by exogenous arachidonic acid

The differentiation of resident tissue macrophages from embryonic precursors and that of inflammatory macrophages from bone marrow cells leads to macrophage heterogeneity. Further plasticity is displayed through their ability to be polarized as subtypes M1 and M2 in a cell culture microenvironment. However, the detailed regulation of eicosanoid production and its involvement in macrophage biology remains unclear. Using a lipidomics approach, we demonstrated that eicosanoid production profiles between bone marrow-derived (BMDM) and peritoneal macrophages differed drastically. In polarized BMDMs, M1 and M2 phenotypes were distinguished by thromboxane B2, prostaglandin (PG) E2, and PGD2 production, in addition to lysophospholipid acyltransferase activity. Although Alox5 expression and the presence of 5-lipoxygenase (5-LO) protein in BMDMs was observed, the absence of leukotrienes production reflected an impairment in 5-LO activity, which could be triggered by addition of exogenous arachidonic acid (AA). The BMDM 5-LO regulatory mechanism was not responsive to PGE2/cAMP pathway modulation; however, treatment to reduce glutathione peroxidase activity increased 5-LO metabolite production after AA stimulation. Understanding the relationship between the eicosanoids pathway and macrophage biology may offer novel strategies for macrophage-associated disease therapy.

a time-response curve ( Supplementary Fig. S4A). Notable, the BMDM cAMP release was 10-fold greater than that PMs under the same experimental conditions ( Supplementary Fig. S4B). Therefore, we next observed the mRNA expression of PGE 2 receptors (EP1, EP2, EP3, and EP4), and LT receptors (BLT1 and BLT2 for LTB 4
However, Ptger2 (EP2) mRNA expression appeared to be up-regulated only in M2s and Ptger3 (EP3) mRNA expression was up-regulated in M1s. In addition, the mRNA expression of BLT1 and BLT2 was up-regulated in both M1 and M2 cells. To assess the basal membrane expression, we used a flow cytometer assay for EP2 and EP4 expression on BMDMs (M0). As showed in Supplementary Fig. S4D, both EP2 and EP4 were expressed although EP4 expression was significantly lower than EP2 expression in the BMDM membrane. (10 ng/mL), IL-13 (10 ng/mL), or IL-4 + IL-13 (10 ng/mL) for 24 h. Total RNA was extracted, synthesized as cDNA, and the relative expression (ΔΔCt) was analysed by qRT-PCR.
Transcripts that encode for (E) sub-type macrophage phenotypes markers, (F) membrane receptors and proteins, and (G) TLR-type receptors and adapter proteins were analysed. The results were normalized to endogenous expression of the internal controls Actb and Gapdh. The
The 2 -ΔΔCt method was used in the analysis of the RT-PCR data.
Cytokine ELISA. Supernatants were harvested and assayed for cytokine content using commercially available ELISA reagents for TNF-α, IL-6, and IL-10 (Duoset R&D Systems, Minneapolis, MN). The detection limit for these cytokines was 7 pg/mL.

Eicosanoid separation and analysis by reverse-phase HPLC coupled to electrospray ionization mass spectrometry (LC-MS/MS). An aliquot of each lipid
sample extracted before (20 µL) was injected into a HPLC column (Accucore C18 -50 x 3 mm, 2.6 µm, Thermo Scientific, EUA) and eluted at a flow rate of 300 µL/min with a linear gradient of HPLC solvent B, which was increased from 45 % to 75 % in 6.5 min, to 98 % in 1 min, and held at 98 % for a further 6.5 min before re-equilibration at 45 % for 10 min. The HPLC system was directly interfaced into the electrospray source of a triple quadrupole mass spectrometer (API 4000, SCIEX, EUA) where mass spectrometric analysis was performed in the negative ion mode using MRM of the following specific m/z transitions (precursor ion → product ion) 6  were injected into an HPLC system connected to a triple quadrupole mass spectrometer (API3200-SCIEX, USA) and normal-phase chromatography was performed using a silica HPLC column (Ascentis, 150 × 2.1 mm, 5 µm, Supelco, Bellefonte, PA) at a flow rate of 200 µL/min. Solvent D was maintained at 25% for 5 min, increased gradually to 60% in 10 min and then to 95% in 5 min, and was held for 20 min before re-equilibration for 15 min. For the enzymatic assay, mass spectrometric analysis was performed in the negative-ion mode using multiple-reaction monitoring (MRM) of the forty-eight molecular species potentially generated during the experimental assay, plus the six deuterated standards 8 . Results are reported as the ratio between the integrated area of each analyte and the integrated area of the corresponding internal standard for each class.
For endogenous phospholipids from BMDM cells, microsomes (10 µg protein), were extracted and analyzed as described above, except that LC-MS/MS analysis was performed using scheduled MRM to detect molecular species containing combinations of common fatty acyl chains 9 . The precursor ions monitored were the molecular ions