Cell culture
Human monocytic THP-1 cells (ATCC TIB-202, Manassas, VA, USA) were seeded in a 12-well plate with 750 000 cells per well and maintained during 24 hours in culture in Roswell Park Memorial Institute (RPMI, Gibco, NY, USA) culture medium containing 10% of heat inactivated fetal bovine serum (Dutscher, Brumath, France), supplemented with 1% penicillin (100 U/mL, Gibco, NY, USA) and streptomycin (0.1 mg/mL, Gibco, NY, USA). THP-1 monocytes are differentiated into M0 macrophages by 24 hours incubation with 100 nM phorbol 12-myristate 13-acetate (P1585, Sigma-Aldrich, St Louis, MO), followed by 24 hours incubation in RPMI medium.
Induction of M1 and M2 Phenotype of THP-1 Cells
Macrophage polarization of THP-1 cells were performed as described with modifications in [14, 15]. Macrophages were polarized in M1 macrophages by 24 hours incubation with 20 ng/mL of human recombinant Interferon ϒ (IFN-γ, 570202 Biolegend, San Diego, CA, USA) and 10 ng/mL of Lipopolysaccharide (LPS) from E.Coli O55:B5 purchased from Sigma-Aldrich. Macrophages M2 polarization was obtained by 72 hours incubation with 20 ng/mL of recombinant human Interleukin 4 (IL-4, 574002, Biolegend, San Diego, CA, USA) and 20 ng/mL of recombinant human Interleukin 13 (IL-13, 571102, Biolegend, San Diego, CA, USA).
LXA4 treatment of polarized macrophages
LXA4 (Cayman Chemical Company, Ann Harbor, MI, USA) was stored at - 80°C until being diluted in serum-free culture medium immediately before use. Once differentiated, M0, M1 and M2 macrophages were treated with 3 different doses of LXA4, 1 nM, 10 nM and 100nM (90410, Bertin Bioreagent, Montigny le Bretonneux, France) or ethanol as a vehicle control.
Cell Viability
THP-1 monocytes were seeded at 750 000 cells/well in 12 well plate and differentiated in macrophages as previously described. Cells were then treated with the different dose of LXA4 for 24 hours and subjected to a CCK-8 cell viability assay (cell counting kit – 8, 96992, Sigma-Aldrich, St Louis, MO). At 24h, cells are rinsed with Phosphate Buffered Saline (PBS, Gibco, NY, USA) and then filled with 440 µL of fresh medium. 40 µL of CCK-8 reagent are added to each well and then left in the incubator at 37°C for 1 hour. 100 µL of supernatant are transferred to a 96 well plate (3 wells per conditions). The absorbance was then measured at 450 nm.
Phagocytosis activity
The functional analysis of the phagocytosis activity of macrophages treated or not with LXA4 was carried out thanks to phagocytosis assays kits (500290, Bertin Bioreagent, Montigny le Bretonneux, France). Once differentiated and treated or not with LXA4, latex beads-rabbit IgG-FITC complex are added in darkness and incubated at 37°C for 4 hours according to the manufacturer’s instructions. M0 macrophages unmarked have served as negative control, as well as M0 macrophages treated with 10 µM cytochalasin D, a phagocytosis inhibitor (C8273, Sigma-Aldrich, St Louis, MO) 30 minutes before addition of latex beads. For all wells, the supernatant is removed and washed with assay buffer. Cells are then removed from the well by gentle scraping with 200 µL of assay buffer and then transferred in a 96-well plate. They are then centrifuged at 2500 rpm for 2 minutes and incubated for 1 minute with trypan blue quenching solution to distinguish cells which have phagocytosed the beads from those simply binding the beads at the surface. Cells are then washed with assay buffer and taken up with 200 µL of PBS in FACS tubes to be analysed by FACS Fortessa X20 (BD Biosciences, San Diego, CA, USA) and FlowJo Software (Tree Star, Ashland, OR, USA).
Surface markers analysis by flow cytometry
Once THP-1 differentiated in M0, M1 and M2, surface markers were evaluated by flow cytometry. Cells were detached by trypsinization, washed with PBS and stained with specific antibody for 30 minutes at 4°C in PBS 0.2% BSA using the following markers: FITC-conjugated anti-CD14 antibody (clone M5E2), APC-conjugated anti-CD80 antibody (clone 2D10), BV711-conjugated anti-HLA-DR antibody (clone L243), PE-conjugated anti-TLR2 antibody (clone W15145C), BV421-conjugated anti-TLR4 antibody (clone HTA125), PE/Dazzle594-conjugated anti-CD163 antibody (clone GHI/61), BV605-conjugated anti-CD206 antibody (clone 15-2), PE/Cy7-conjugated anti-CD86 antibody (clone IT2.2,) and APC/Cy7-conjugated ZombieNIR to check the viability of cells (all by Biolegend, San Diego, CA, USA). Cells were washed twice and transferred in 200 µL of PBS in FACS tubes. Cells were analysed by FACS Fortessa X20 (BD Biosciences, San Diego, CA, USA) and FlowJo Software (Tree Star, Ashland, OR, USA).
Bead based immunoassay LEGENDplexTM
Bead based multiplex assay (LEGENDplexTM human macrophages/microglia panel, Biolegend, San Diego, CA, USA) was used to measure the secretion of cytokines after macrophages polarization. The protocol was performed per the manufacturer’s instructions. Briefly, detection beads were vortexed and incubated with supernatant of polarized THP-1. Beads bound to target cytokines were then washed, incubated with detection antibodies, washed, incubated with streptavidin-phycoerythrin which will bind to the biotinylated detection antibodies, washed again and finally the cytokine concentrations were determined through flow cytometry analysis. It allows for simultaneous quantification of 13 key targets involved in monocytes differentiation and macrophage functions including IL-10, IL-12p70, IL-12p40, IL-1RA, IL-1β, IL-23, IL-6, IP-10, TARC and TNF-α. Data were analysed via LegendplexTM software and specified as pg/mL.
RTqPCR
To validate macrophage polarization, real time PCR was used to measure the expression levels of genes encoding specific markers of macrophage phenotype. After differentiation with IFN-γ, LPS and IL-4, IL-13, total RNA was extracted from M0, M1 and M2 macrophages using NucleoSpin RNA extraction kit (Macherey-Nagel, Düren, Germany) according to manufacturer’s instructions. In the same way, total RNA was extracted from M0, M1 and M2 macrophages treated or not with LXA4. 1 µg quantity of total RNA was reverse transcribed (Verso cDNA synthesis kit, ThermoFisher Scientific Saint-Aubin, France). Real time PCR was performed using the SyBR Select Master Mix (Applied Biosystems) and analysed using a Bio-Rad CFX96 detection system. The cycling conditions were as follow: 50°C 2 minutes; 95°C 2 minutes; 40 cycles of 95°C 15 seconds, 60°C 30 seconds. Specificity of the amplified products was confirmed by analysing the melting curves. The mRNA expression levels were normalized to that of housekeeping genes (B2M, PPIA) and calculated using the 2-ΔΔCT method. The following primers have been used from EUROFINS (MWG Operon, Nantes, France): TNF-αfor 5’- CAGCCTCTTCTCCTTCCTGAT-3’, TNF-αrev 5’-GCCAGAGGGCTGATTAGAGA-3’ (NM000594) IL-6for 5’- TCCACAAGCGCCTTCGGCCAG-3’, IL-6rev 5’-CTCAGGGCTGAGATGCCGTCG-3’ (NM_000600.5), IL-1βfor 5’ CCGGGACTCACAGCAAAA-3’, IL-1βrev 5’-GGACATGGAGAACACCACTTG-3’ (NM 000576), CD206for 5’-AGCCAACACCAGCTCCTCAAGA-3’, CD206rev 5’-CAAAACGCTCGCGCATTGTCCA-3’ (NM_002438.4), FPR2for 5’- GAGCTTAGCTGCTGGTGCT-3’, FPR2rev 5’-CACCCAGATCACAAGCCCAT-3’ (NM_001005738.2), B2Mfor 5’- CCTGGAGGCTATCCAGCGTA-3’, B2Mrev 5’-GGATGACGTGAGTAAACCTG-3’ (NM_004048.4), PPIAfor 5’- GTCAACCCCACCGTGTTCTT-3’, PPIArev 5’- CTGCTGTCTTTGGGACCTTGT-3’ (NM_021130.5), 18Sfor 5’-AGCAAACCCCAACTCAACC-3’, 18Srev 5’ GTCCCTCAGAAGGGGTGAC-3’ (NM_018135.4), and from Qiagen (Germantown, MD, USA) QT00000364 (NM_002187) (IL-12), QT00041685 (NM_000572) (IL-10).