Modified Vaccinia Virus Ankara Triggers Type I IFN Production in Murine Conventional Dendritic Cells via a cGAS/STING-Mediated Cytosolic DNA-Sensing Pathway
Figure 1
MVA induces type I IFN production in conventional dendritic cells (cDCs).
(A) Murine pDCs and cDCs were purified from Flt3L-BMDCs using FACS. 2×105 pDCs (CD11c+B220+PDCA-1+) and 1×106 cDCs (CD11c+B220−PDCA-1−) were either stimulated with CpG (at a final concentration of 10 µg/ml) or infected with either WT VAC or MVA at a MOI of 10. Supernatants were collected at 22 h post infection. The concentrations of IFN-α and IFN-β were determined by ELISA. Data are means ± SEM (n = 6). A representative experiment is shown, repeated at least twice. (B) GM-CSF-BMDCs (1×106) were infected with WT VAC or MVA at a MOI of 10. Supernatants were collected at 1, 4, 8, 14, and 22 h post infection. The concentrations of IFN-α and IFN-β were determined by ELISA. Data are means ± SEM (n = 3). A representative experiment is shown, repeated once. (C) GM-CSF-BMDCs (1×106) were infected with MVA at a MOI of 0.25, 0.5, 1, 5, or 10. Supernatants were collected at 22 h post infection. The concentrations of IFN-α and IFN-β were determined by ELISA. Data are means ± SEM (n = 3). A representative experiment is shown, repeated once. (D) GM-CSF-BMDCs (1×106) were infected with MVA or WT VAC at a MOI of 10. Cells were collected at 6 h post infection. Real-time PCR analysis of IFNA4 and IFNB mRNAs were performed. Data are means ± SEM (n = 3). A representative experiment is shown, repeated twice. ***, p<0.001; comparisons were made between MVA and WT VAC infected cells. (E) GM-CSF-BMDCs (1×106) were infected with MVA or WT VAC at a MOI of 10. Cells were collected at 1, 2, 4, and 8 h post infection. Western blot analysis was performed using anti-phospho-TBK1, anti-TBK1, anti-phosphoserine-396 of IRF3, and anti-IRF3. Glyceraldehyde 3-Phosphate Dehydrogenase (GAPDH) was used as a loading control. “hpi”, hours post infection. “M”, mock infection control.