2.2. In vitro generation of monocyte-derived DC

Individual donor monocyte preparations outlined above were used to generate the different DC phenotypes for each experiment. Each donor therefore acted as a replicate for each experiment, with associated intra-donor variability. For each donor, CD14+ monocytes were differentiated into DC in vitro according to the schematic shown in Fig. 1A. Monocytes were cultured for 5 days with granulocyte macrophage-stimulating colony factor (GM-CSF; 800U/mL, Berlex Laboratories, Seattle, WA) and Interleukin-4 (IL-4; 400U/mL), in the presence or absence of 10 nM 1,25D (Enzo Life Sciences, Exeter, UK; diluted in RPMI 1640 medium from 50 μg/mL stock), at 37 °C and 5% CO₂. Fresh medium supplemented with GM-CSF and IL-4 was added on day 2 and day 5 of culture, with the resulting day 6 cells being immature DC (iDC). The addition of 1 μg/mL LPS (from E. coli, Sigma Aldrich) for 24 h on day 6 generated mature DC (mDC). Addition of 1,25D (10 nM) for all 6 days of culture in the absence of LPS was used to generate immature tolerogenic DC (itolDC) and the addition of LPS for the last 24 h of these cultures generated mature tolerogenic DC (mtolDC). In some cultures mitochondrial fatty acid synthase (FAS) was specifically inhibited using 25 μM of 4-Methylene-2-octyl-5-oxotetrahydrofuran-3-carboxylic acid (C75, Sigma Aldrich) from day 0.

Regulation of DC phenotype by 1,25D. A. Schematic representation of the model for cell culture of human monocyte-derived DC. Immature DC (iDC), mature DC (mDC), immature tolerogenic DC (itolDC), mature tolerogenic DC (mtolDC). B. Representative flow cytometry analyses for CD11c, CD14, CD80, CD86 and HLA-DR in iDC, mDC, mtolDC and itolDC showing comparison between iDC vs itolDC, iDC vs mDC and mDC vs mtolDC. C. Median fluorescence intensity values for CD11c, CD14, CD40, CD80, CD83, CD86, CD209 and HLA-DR in iDC, mDC, mtolDC and itolDC. D. Expression of mRNA for VDR, CYP24A1 and CYP27B1 in iDC, mDC, mtolDC and itolDC. Data are shown as: raw delta Ct values with associated statistical analysis (upper panel); fold-change in mRNA expression (lower panel). Data show individual replicate cell preparation values and median and upper/lower quartiles for n = 5 replicate donor analyses. * = statistically different from iDC values, p < 0.05, ** p < 0.01, *** p < 0.001.