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.
Figure 2.
Transcription factors IRF3/IRF7 and the type I IFN positive feedback loop mediated by IFNAR1 are required for the induction of type I IFN in murine cDCs by MVA.
GM-CSF-BMDCs were generated from IRF3−/− (A), IRF7−/− (B), IFNAR1−/− (C) mice, and their age-matched WT controls. Cells (1×106) were stimulated with CpG or infected with MVA at a MOI of 10. Supernatants were collected 22 h later. The concentrations of IFN-α and IFN-β were determined by ELISA. Data are means ± SEM (n = 3). A representative experiment is shown, repeated twice. *, p<0.05; **, p<0.01; ***, p<0.001; comparisons were made between WT cells and various knockout cells as indicated.
Figure 3.
TLR9 and MyD88 contribute to the induction of type I IFN in cDCs by MVA.
GM-CSF-BMDCs were generated from MyD88−/− (A), TLR9−/− (B), TLR7−/− (C) mice, and their age-matched WT controls. Cells (1×106) were either stimulated with CpG or infected with MVA at a MOI of 10. Supernatants were collected 22 h later. The concentrations of IFN-α and IFN-β were determined by ELISA. Data are means ± SEM (n = 6). The combined results of three independently performed experiments are shown. *, p<0.05; **, p<0.01; ***, p<0.001; comparisons were made between WT cells and various knockout cells as indicated.
Figure 4.
STING is required for the induction of type I IFN and IRF3 phosphorylation by MVA in BMDCs.
GM-CSF-BMDCs were generated from WT mice (Sting+/+) and the N-ethyl-N-nitrosourea (ENU)-induced Goldenticket (Gt) mutant mice (StingGt/Gt) harboring a single nucleotide variant of Sting resulting in a functionally null allele. (A) Cells (1×106) were either stimulated with LPS or infected with MVA at a MOI of 10. Supernatants were collected 22 h later. The concentrations of IFN-α and IFN-β were determined by ELISA. Data are means ± SEM (n = 3). Results shown are representative of three experiments. ***, p<0.001; comparisons were made between Sting+/+ and StingGt/Gt as indicated. (B) Cells (1×106) were infected with MVA 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<0.01; ***, p<0.001; comparisons were made between Sting+/+ and StingGt/Gt as indicated. (C) Western blot analysis of BMDCs from Sting+/+ and StingGt/Gt infected with MVA at a MOI of 10, or mock infected. Whole-cell lysates were prepared. Equal amount of proteins were subjected to SDS-PAGE and immunoblotting with anti-phospho-TBK1, anti-TBK1, anti-phosphoserine-396 of IRF3, and anti-IRF3. GAPDH was used as a loading control. “hpi”, hours post infection. “M”, mock infection control. (D) StingGt/Gt, IRF3−/− and age-matched WT C57B/6 control mice were infected with MVA (2×107 pfu) via intravenous inoculation. Serum levels of IFN-α and IFN-β were determined by ELISA. Data are means ± SD. Results shown are representative of two independent experiments.
Figure 5.
cGAS is the critical cytosolic DNA sensor for MVA infection of cDCs.
GM-CSF-BMDCs were generated from cGAS−/− mice and its age-matched WT controls. (A) Cells (1×106) were infected with MVA at a MOI of 10. Supernatants were collected 22 h later. The concentrations of IFN-α and IFN-β were determined by ELISA. Data are means ± SEM (n = 3). A representative experiment is shown, repeated twice (***, p<0.001). (B) Cells (1×106) were infected with MVA 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). (C) Western blot analysis of cGAS+/+ and cGAS−/− cDCs infected with MVA at a MOI of 10, or mock infected. Whole-cell lysates were prepared. Equal amount of proteins were subjected to SDS-PAGE and immunoblotting with anti-phospho-TBK1, anti-TBK1, anti-phosphoserine-396 of IRF3, and anti-IRF3. GAPDH was used as a loading control. “hpi”, hours post infection. “M”, mock infection control.
Figure 6.
Viral DNA replication is not required for MVA-induced IRF3 phosphorylation.
(A) GM-CSF-BMDCs (1×106) were pre-incubated with either PAA at 200 µg/ml or with mock control for 1 h, and then infected with MVA at a MOI of 3 for 1 h. Cells were washed and incubated with fresh medium with or without PAA. Cells were collected at 1, 4, 8, and 24 h post infection. Viral DNA was extracted and purified. Real-time PCR was performed with primers and TagMan probe specific for the vaccinia ribonucleotide reductase l4L gene. (B) Western blot analysis of BMDCs infected with MVA at a MOI of 10 in the presence of absence of PAA. Whole-cell lysates were prepared. Equal amount of proteins were subjected to SDS-PAGE and immunoblotting with anti-phosphoserine-396 of IRF3 or anti-IRF3. Glyceraldehyde 3-Phosphate Dehydrogenase (GAPDH) was used as a loading control. “hpi”, hours post infection. “M”, mock infection control.
Figure 7.
Endosomal and lysosomal acidification and lysosomal enzyme activities are required for type I IFN production by BMDCs in response to MVA infection.
(A) GM-CSF-BMDCs (1×106) were infected with MVA at a MOI of 10 for 1 h. Cells were washed and incubated in fresh medium in the presence or absence of chloroquine (50 µM), bafilomycin A1 (100 nM), or CA-074-Me (10 µM). Supernatants were collected 22 h later. The concentrations of IFN-α/β were determined by ELISA. Data are means ± SEM (n = 6). The combined results of three independently performed experiments are shown. **, p<0.01; ***, p<0.001; compared with no drug treatment. (B) GM-CSF-BMDCs were generated from Cathepsin B KO (CathB−/−) and age-matched WT controls (CathB+/+). Cells (1×106) were infected with MVA at a MOI of 10. Supernatants were collected 22 h later. The concentrations of IFN-α/β were determined by ELISA. Data are means ± SEM (n = 3). A representative experiment is shown, repeated twice. **, p<0.01; comparisons were made between CathB+/+ and CathB−/− cells as indicated.
Figure 8.
MVAΔE3L induces high levels of type I IFN gene expression in BMDCs than MVA does.
(A) GM-CSF-BMDCs were generated from 6–8 week-old female WT C57B/6 mice. Cells (1×106) were infected with MVA or MVAΔE3L 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 MVAΔE3L infected cells. (B) GM-CSF-BMDCs were generated from IRF3−/− mice and age-matched WT C57B/6 mice. Cells were infected with MVA or MVAΔE3L at a MOI of 10, or mock infected. 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<0.01; ***, p<0.001; comparisons were made between IRF3+/+ and IRF3−/− cells. (C) Western blot analysis of GM-CSF-BMDCs infected with MVA or with MVAΔE3L at a MOI of 10, or mock infected. Whole-cell lysates were prepared. Equal amount of proteins were subjected to SDS-PAGE and immunoblotting with anti-phosphoserine-396 of IRF3. β-actin was used as a loading control. “hpi”, hours post infection. “M”, mock infection control.
Figure 9.
Vaccinia N1 virulence factor plays an inhibitory role in the type I IFN gene induction.
GM-CSF-BMDCs (1×106) were infected with MVA or MVA-N1L at a MOI of 10. (A) Cells were collected at 6 h post-infection. Real-time PCR analysis of IFNA4 and IFNB mRNAs were performed. Data are means ± SEM. A representative experiment is shown, repeated twice. (B) Western blot analysis of GM-CSF-BMDCs infected with MVA or MVA-N1L at a MOI of 10. Whole-cell lysates were prepared. Equal amount of proteins were subjected to SDS-PAGE and immunoblotting with anti-phospho-TBK1, anti-TBK1, anti-phosphoserine-396 of IRF3 and anti-IRF3. GAPDH was used as a loading control. “hpi”, hours post infection. “M”, mock infection control. (C) Cells (1×106) were infected with MVA or MVA-N1L at a MOI of 10. Supernatants were collected 22 h later. The concentrations of IFN-α and IFN-β were determined by ELISA. Data are means ± SEM (n = 3). A representative experiment is shown, repeated twice (**, p<0.01).