Figure 4.

Vitamin-D derivatives induce LL37 mediated Leishmania restriction in human primary macrophages. hMDM-1 were generated and treated with calcitriol (CCT) or calcipotriol (CPT) for 24 h, or left untreated. Subsequently, hMDM-1 were infected with transgenic dsRed expressing Lm or Lae (MOI10). After 3 h, extracellular parasites were removed by washing, following incubation at 37°C, 5% CO₂. After 24 h (early; black bars) or 6 d (late; white bars) CAMP gene (n = 1–5) (A) and cathelicidin protein expression (n = 5–7) (B) were by qRT-PCR and western blot analysis. CAMP expression was normalized against the house keeping gene GAPHD. Western blots were analyzed by densitometry (ImageJ analysis), normalizing cathelicidin against ß-actin protein expression. Gene and protein expression were normalized to the untreated control, which was set to 1 (dashed line). In addition, parasite survival (C,D) and infection rates of Lm dsRed (E), analyzed by the mean fluorescent intensity (MFI) of the dsRed protein, was assessed by flow cytometry. Western blots, FACS histograms and data, presented as mean ± SD, are representative for at least 3 independent experiments (Wilcoxon matched-pairs signed rank test; *p < 0.05; **p < 0.01).