Monocyte Isolation and Differentiation

Peripheral blood mononuclear cells (PBMCs) from healthy donors (Karolinska Hospital Blood Bank, Stockholm, Sweden) were isolated from buffy coat blood by density sedimentation over Lymphoprep (GE Healthcare Life Sciences, Logan, UT). Cells were allowed to adhere in 6-well culture plates (Nunc, NY, Rochester, USA) for 2 h at 37°C in serum free media (RPMI 1640, supplemented with 2 mM L-glutamine and 5 mM HEPES (Hyclone, GE Healthcare Life Sciences). The non-adherent cells were removed by washing with phosphate buffered saline (PBS) and thereafter adherent monocytes were differentiated for 6 days in cell culture media containing 10% of fetal calf sera (FCS) (Sigma-Aldrich, St. Louis, MO). To establish an unbiased culture system that could assess the individual effects of 1,25(OH)₂D₃ compared to conventional M1 and M2 polarization methods, unstimulated control (M0) and 1,25(OH)₂D₃-polarized monocytes were differentiated in the absence of external growth factors using similar protocols as previously described (21, 22). As the maturation and activation status of macrophages differs depending on the in vitro stimulant (23), the cells obtained with the differentiation protocols used are referred to as monocyte-derived cells. Briefly, cells were left unstimulated or conditioned with 50 ng/mL human M-CSF (Life technologies, Carlsbad, CA) or 50 ng/mL human GM-CSF (ImmunoTools, Germany) for 3 days, after which the media was replaced with fresh media with or without M-CSF or GM-CSF (100 g/ml) for another 3 days of culture. To obtain 1,25(OH)₂D₃-polarized monocyte-derived cells or fully polarized M1 or M2 subsets, unstimulated monocyte cultures were treated with 10 nmol vitamin D [1,25(OH)₂D₃, Sigma-Aldrich], while GM-CSF differentiated monocytes were treated with 50 ng/mL interferon-gamma (IFN-γ) and 10 ng/mL lipopolysaccharide (LPS), and M-CSF differentiated monocytes were treated with 20 ng/mL IL-4 (ImmunoTools, Germany), 18–20 h prior to infection with Mtb. The macrophage polarization protocols (8) and nomenclature used in this study is summarized in Table 1. The morphology of monocyte-derived cell cultures was monitored with light microscopy and the viability of adherent cells after 7 days of culture was estimated to >95% using Trypan Blue (Sigma-Aldrich) staining.

Polarization of monocyte-derived cells.

We used a fixed dose of 1,25(OH)₂D₃ (10 nmol) that was in a similar range as the doses previously used by us (18, 24) and other groups (17, 19) to study the in vitro effects of 1,25(OH)₂D₃ on human immune cells. The circulating 25(OH)D₃ proform (10–100 nmol/L in serum) is converted to equivalent concentrations of the active 1,25(OH)₂D₃ form inside cells upon uptake of 25(OH)D₃ (20), which is the basis for using the active form in the nanomolar range in cellular experiments, corresponding to a physiological concentration of active vitamin D.