Monensin

Cell culture medium for PBMCs and DCs was RPMI 1640 (Gibco, Carlsbad, CA, USA) supplemented with 10% FCS (Gibco, Carlsbad, CA, USA) and 100 U/mL streptomycin/penicillin. Anti-human CD3, anti-human CD28, anti-human CD4-FITC, anti-human CD25-APC, anti-human Foxp3-PE, anti-human IFN-γ-PE, anti-human IL-17-PE, anti-human CD11c-APC/Cy7, anti-human HLA-DR-PerCp/Cy5.5, anti-human CD40-PE, and anti-human CD86-PE were all from eBioscience, San Diego, CA, USA. TRIzol, PrimeScript RT reagent kit, and SYBR Green Master Mix were from Takara Biotechnology, Dalian, China. Recombinant human GM-CSF and recombinant human IL-4 were obtained from PeproTech, Rocky Hill, NJ, USA. Meanwhile, human magnetic CD14 isolation kit and CD4 isolation kit were bought from Miltenyi Biotec, Auburn, CA, USA. Lipopolysaccharides (LPS) were from Sigma-Aldrich, St Louis, MO, USA, and recombinant human IL-37 was bought from AdipoGen AG, Liestal, Switzerland. Oxidised low density lipoprotein (oxLDL) was acquired from Yiyuan, Wuhan, China, and phorbol myristate acetate, ionomycin, and monensin were from Alexis Biochemicals, San Diego, CA, USA.

To analyze the prevalence of Tc17 cells, IL-17-producing CD8(+) lymphocytes were evaluated by flow cytometry. In brief, heparinized peripheral whole blood (200 µl) with an equal volume of RPMI-1640 medium was incubated for 4 h at 37°C in 5% CO₂ in the presence of 25 ng/ml of phorbol myristate acetate (PMA), 1 µg/ml of ionomycin, and 1.7 µg/ml monensin (all from Alexis Biochemicals, San Diego, CA). Then, the cells were incubated with PE-Cy5-conjugated anti-human CD3 and FITC-conjugated anti-human CD8 monoclonal antibodies (eBioscience, San Diego, CA) at room temperature in the dark for 15 min to stain the surface. After fixation and permeabilization, according to the manufacturer's instructions, the cells were stained with a PE-conjugated anti-IL-17 monoclonal antibody for 15 min. Isotype controls were used to enable correct compensation and confirm antibody specificity. Stained cells were analyzed by flow cytometric analysis using a FACScan cytometer equipped with CellQuest software (BD Bioscience PharMingen).

Flow cytometry was used to study membrane makers and intracellular cytokines to identify the cytokine-producing cells. Briefly, heparinized peripheral blood with an equal volume of 1640 medium (Hyclone, USA) was incubated for 4 h at 37°C, 5% CO2 in the presence of 25 ng/ml of phorbol myristate acetate (PMA), 1 μg/ml of ionomycin, and 1.7 μg/ml monensin (all from Alexis Biochemicals, USA). After incubation, the cells were stained with Alexa Fluor^® 647 or PerCP/Cy 5.5 anti-CD4 monoclonal antibody at room temperature in the dark for 20 min. After surface staining, the cells were next stained with FITC anti-IFN-γ, PerCP/Cy 5.5 or PE anti-IL-17 and PE or Alexa Fluor^® 660 anti-IL-22 monoclonal antibodies after fixation and permeabilization. All the antibodies were purchased from Biolegend and eBioscience (California, USA). Fixation and permeabilizaton reagents were purchased from eBioscience (California, USA). Isotype controls were given to enable correct compensation and confirm antibody specificity. Stained cells were analyzed by flow cytometric analysis using a FACS Calibur cytometer equipped with CellQuest software (BD Bioscience PharMingen, USA). For analysis, we first gated lymphocytes, then gated CD4⁺IFN-γ⁻ T cells in lymphocytes, and analyzed the percentages of CD4⁺IFN-γ⁻IL-17⁻IL-22⁺ (Th22) cells in CD4⁺IFN-γ⁻ T cells.

For analysis of Treg cells, PBMCs were surface-labeled with CD4-PE/Cy5, CD25-PE followed by fixation and permeabilization and intracellularly stained with Foxp3-Alexa Fluro488 or were surface-labeled with CD4-PE/Cy5, CD25-PE, and CD127-FITC (eBioscience, USA).
For analysis of Th17 cells, PBMCs was suspended in complete culture medium (RPMI1640 was supplemented with 10% heat-inactivated fetal calf serum, (Gibco BRL, USA)). Cultures were stimulated for 1 hour using 50 ng/mL phorbol myristate acetate (PMA) and 1 μg/mL ionomycin (Sigma-Aldrich, USA) and then stimulated using 500 ng/mL monensin (Alexis Biochemicals, USA) for 3 hours at environment of 37°C and 5% CO₂. Cells were then washed in PBS and surface-labeled with CD4-PE/Cy5 (eBioscience, USA). Following surface staining, cells were fixed and permeabilized using fixation/permeabilization buffer (eBioscience, USA) and then stained with IL-17a-PE (eBioscience, USA). Labeled cells were washed and analyzed with FACS Calibur flow cytometer (Becton-Dickinson, USA) using FlowJo (Tree Star, USA) v7.6.1 software. In each case, staining was compared with that of the appropriately labeled isotype control antibody.