Lipopolysaccharides (lps)

After 7 days of differentiation, cells were washed twice with sterile PBS. For M1-activation, 2 mL RPMI 1640/5% FCS containing 1 μg/mL LPS (Sigma-Aldrich Chemie GmbH, Munich, Germany) + 300 U/mL IFNy (Miltenyi Biotec, Bergisch Gladbach, Germany) were added to the wells. M2-activation was induced by the addition of medium containing 40 ng/mL IL-13 (Miltenyi Biotec, Bergisch Gladbach, Germany) + 40 ng/mL IL-4 (Miltenyi Biotec, Bergisch Gladbach, Germany) instead of LPS + IFNy. Control cells were cultured with RPMI 1640/5% FCS medium only. Cells were then incubated for 24 h under these conditions as described by Mosser and Zhang [26 (link)]. In order to demonstrate successful macrophage polarization, culture samples were stained with anti-CD206 (to assess M2-polarization) and anti-iNOS (to assess M1 polarization). Representative histograms are shown in Supplementary Figure  1 in Supplementary Material available online at https://doi.org/10.1155/2017/7584621.

CD14+ monocytes were purified by means of cell sorting and were left untreated or were pretreated with URB597 for 30 min. To allow cytokine synthesis, the monocytes were then stimulated with 100 ng/mL of LPS (Sigma-Aldrich, St. Louis, MO, USA) for 5 h in the presence of 10 μg/mL of brefeldin A (Life Technologies, Carlsbad, CA, USA) and cytokine production was evaluated by flow cytometry, as reported [17 (link)]. For macrophage polarization, the CD14+ monocytes were left in adhesion for 2 h in complete RPMI 1640 medium in order for them to adhere. After two hours, non-adherent cells were removed and adhering monocytes were gently rinsed with PBS and cultured in fresh complete medium supplemented with 50 ng/mL of M-CSF (Miltenyi Biotec, Bergisch Gladbach, Germany) for M0 (homeostatic macrophages) polarization for 6 days. At day 2 and 4, cells were provided with new supplemented medium, as reported [18 (link)]. At day 6, cells were rinsed with PBS and polarized into M1 (pro-inflammatory macrophages) in the presence of 100 ng/mL of LPS and 10 ng/mL of mouse IFN-γ (Miltenyi Biotec, Bergisch Gladbach, Germany). Adherent M1 macrophages were collected following the addition of trypsin-EDTA solution, stained and analyzed for immunophenotyping by flow cytometry, as detailed in Section 2.4.

Macrophage polarization was obtained by supplementing the medium with 100 ng/mL LPS and 20 ng/mL IFN-γ (Miltenyi Biotec) for M1-like cells and with 20 ng/mL IL-4 (Miltenyi Biotec) for M2-like cells. For this, macrophages were seeded at a concentration of 95,000 cells/cm² in 24-wells plates for 24 h. CD68 (Cluster of Differentiation 68) staining was performed by incubating fixed macrophages with a 1:100 dilution of the mouse monoclonal antibody (ab955 from Abcam, Cambridge, United Kingdom) in a solution of 0.1% bovine serum albumin (BSA) in PBS at 4 °C overnight. On the following day, the secondary anti-body Alexa Fluor 488 conjugate (Invitrogen, Carlsbad, USA) was incubated for 1 h as a 1:500 dilution. 4′,6-diamidino-2-phenylindole (DAPI) was used to counterstain cell nuclei, supplemented as a 1:1000 solution in 0.1% BSA. All supplements were purchased from Sigma-Aldrich unless stated otherwise. CD68 stainings included 3 independent experiments with a minimum of 2 technical replicates each.

Human monocytes were obtained from 6 healthy volunteers. Human peripheral blood mononuclear cells (PBMC) were isolated from venous blood by density gradient centrifugation on Lympholyte Cell Separation Media (Cedarlane Laboratories Limited, Burlington, ON, Canada). CD14⁺ monocytes were separated from PBMCs by immunomagnetic sorting using anti-CD14 (MACS CD14 Microbeads; Miltenyi Biotec, Auburn, CA, USA) magnetic microbeads [21 (link)]. Immunomagneting sorting efficiency was 98% according to flow cytometry analysis (data not shown). CD14⁺ monocytes were immediately subjected to macrophage differentiation, as described by Tarique and co-workers [22 (link)]. Briefly, CD14⁺ monocytes were cultured in RPMI 1640 medium (Sigma-Aldrich, USA) supplemented with 10% FBS, 1% penicillin/streptomycin (Sigma-Aldrich, USA), 10 mM HEPES (Euroclone, Pero MI, Italy) and 1 mM L-glutamine (Lonza, USA). M1-like Macrophage differentiation was performed with 50 μg/mL GM-CSF (Miltenyi Biotec, Auburn, CA, USA) for 5 days, followed by 4 days of 20 μg/mL LPS + 20 μg/mL IFNγ (Miltenyi Biotec, Auburn, CA, USA) treatment. M0-like macrophages were obtained after 50 μg/mL GM-CSF treatment for 9 days, while M2-like macrophages were differentiated with 50 μg/mL GM-CSF (Miltenyi Biotec, Auburn, CA, USA) for 5 days, followed by 20 μg/mL IL-4 + 20 μg/mL IL-13 (Miltenyi Biotec, Auburn, CA, USA) treatment for 4 days.