Recombinant porcine il 4

Porcine blood monocyte-derived dendritic cells (MoDCs) were generated by a five-day protocol as previously described by [20 (link)]. Briefly, porcine monocytes were obtained from freshly collected porcine peripheral blood by a specific gravity centrifugal method with Lympholyte-mammal (CEDARLANE, HornBy, Ontario, Canada). Cells were cultured in complete RPMI1640 culture medium containing: 2 mM of l-glutamine, 100 U/ml of Polymixin B, 10 % of FCS, 100 ug/ml of penicillin, 100 U/ml of streptomycin, 20 ng/ml of recombinant porcine GM-CSF (R & D Systems), and 20 ng/ml of recombinant porcine IL-4 (R & D Systems). Cells were cultured at a density of 5.0 × 10⁶ cells/well (12-well plate), or 2.0 × 10⁷cells/well (6-well plate) at 37 °C in 5 % CO₂ atmosphere. Cells were incubated for 5 days with cytokine-containing medium that was replaced on day 3. These cells are referred as immature MoDcs. For the obtainment of mature DCs, LPS (1 μg/ml, L2630 Sigma, lipopolysaccharides from Escherichia coli 0111:B4) was added to the medium on day 3. Mature MoDCs were harvested on day 5 or 7 using cell dissociation enzyme-free Hank’s-based buffer (Gibco® Cell Dissociation Buffer), and suspended in complete RPMI1640.

Peripheral blood samples were collected into 50 mL centrifuge tubes with 0.5% heparin sodium, diluted in a ratio of 1:2 with sterile 1×PBS, overlaid on Ficoll-Hypaque (TBD science, Tianjin, China) and PBMCs were collected as previously detailed [32 (link)]. CD14⁺ monocytes were purified from PBMCs by immunomagnetic labeling of cells using mouse anti-pig CD14 monoclonal antibody (AbD Serotec, Raleigh, NC, USA) and anti-mouse IgG MicroBeads (Miltenyi Biotec, Bergisch-Gladbach, Germany) according to the manufacturer’s protocol. The CD14⁺ monocytes were cultured for 5 days in RPMI-1640 medium supplemented with 10% fetal bovine serum (FBS), 25 ng/mL recombinant porcine IL-4 and 10 ng/ml recombinant porcine GM-CSF (R&D systems, Minneapolis, MN, USA). The cell culture medium was replenished every 3 days. The typical veiled morphology of the cells was checked for every day. After five days of culture, CD1, SLA-II, and CD172a markers on these cells were analyzed by flow cytometry using monoclonal antibodies [33 (link), 34 (link)].

Mφ cultures were obtained from blood leukocytes, as previously described [21 (link)]. Leukocytes were cultured for 7 days in RPMI-1640 supplemented with 10% fetal bovine serum (FBS), 100 U/mL streptomycin, and 100 μg/mL penicillin (complete RPMI, cRPMI), and with 50 ng/mL of recombinant human M-CSF (hM-CSF) (Thermo Fisher Scientific, Waltham, MA, USA), using Petri dishes, as previously described [21 (link)]. MoMΦ were then harvested, washed, re-suspended in cRPMI and seeded in 12-well plates (Greiner CELLSTAR, Sigma-Aldrich, Saint Louis, MO, USA) (8–10 × 10⁵ live cells per well) or 8-well chamber slides (Nunc Lab-Tek chamber slide system, Sigma-Aldrich) (1 × 10⁵ live cells per well). Cells were incubated at 37 °C 5% CO₂ for further 24 h before stimulation. moMΦ were left untreated or stimulated with recombinant porcine IL-10 or TGF-β: culture medium was replaced with fresh cRPMI containing either IL-10 (20 ng/mL) or TGF-β (20 ng/mL) (both R&D Systems, Minneapolis, MN, USA). In defined experiments, culture medium was instead replaced with fresh cRPMI containing LPS (lipopolysaccharide from Escherichia coli 0111:B4; Sigma-Aldrich) (1 μg/mL) or recombinant porcine IL-4 (20 ng/mL) (R&D Systems), which were used as positive controls.