1α,25(OH)₂D₃ inhibits the listericidal activity in IFN-γ–activated macrophages. (A) BMDMs were cultured in the presence or absence of IFN-γ and 1α,25(OH)₂D₃ for 24 hours and infected with opsonized L monocytogenes (MOI = 0.1) for 1 hour and 3 hours. Extracellular growth of L monocytogenes was prevented by the addition of gentamicin to the medium and intracellular bacteria were quantified by counting the number of CFUs in the cell lysates on BHI plates. (B) BMDMs were cultured in the presence or absence of IFN-γ and 1α,25(OH)₂D₃ for 48 hours and infected and analyzed as described in panelA. The data are representative of at least 5 independent experiments. Similar results were obtained when nonopsonized L monocytogenes was used in the infection experiments. (C) BMDMs were cultured with IFN-γ and different concentrations of 1α,25(OH)₂D₃ and infected with L monocytogenes for 3 hours. (D) BMDMs were cultured with IFN-γ and 1α,25(OH)₂D₃ and infected with L monocytogenes mutants deficient for listeriolysin (del hly). Experiments were repeated twice with similar results (A,C-D). Data are depicted as the mean ± SEM calculated from triplicate wells (plating was carried out in duplicate). ^*P < .05; Wilcoxon-signed rank test. VitD₃ indicates 1α,25-dihydroxycholecalciferol. IFN-γ = 500 U/mL, VitD₃ = 40 nM, and treatment was performed for 48 hours except as otherwise indicated.