TNF-α protein secretion from LPS-induced Mφ and non-stimulated Mφ pre-treated with 25(OH)D₃ and 1.25(OH)₂D₃ for T = 12 h and T = 24h.

Mφ (n = 6) were pre-treated with either 25(OH)D3 (100 nM and 500 nM) or 1.25(OH)2D3 (0.05 nM and 10 nM) for (a) 12 hours followed by LPS challenge (1 μg/mL) for 4 hours or (b) 24 hours followed by LPS challenge (1 μg/mL) for 4 hours. (c) for 12 hours and (d) for 24 hours. TNF-α protein levels (pg/mL) were measured in Mφ culture medium by Enzyme-linked immunosorbent assay (ELISA). Repeated measures One-way ANOVA Test of trend was performed to evaluate dose-dependent response of either 25(OH)D3 or 1.25(OH)2D3. P-value numbers are stated over the specific groups. One-way ANOVA analysis and Dunnett’s multiple comparisons test was also performed to compare the means of control group with each stimulation group and significance in illustrated as *. (a) * Significant difference between LPS-induced Mφ and Mφ pre-treated with 10 nM 1.25(OH)2D3. (b) ** Significant difference between LPS-induced Mφ and Mφ pre-treated with 500 nM 25(OH)D3 and 10 nM 1.25(OH)2D3. (d) * Significant difference between control Mφ and Mφ pre-treated with 500 nM 25(OH)D3.