SUPPLEMENTAL FIGURE 1
(A) IL-6, IL-10 and IFN-α in supernatants from J774 stimulated for 24 h with CpG,
R848, 3’-triphosphate RNA complexed with Lipofectamine (3P-RNA) or poly(I:C) complexed with Lipofectamine (p(I:C)+Lipo).
(B) IL-6, IL-12p40 and IL-12p70 in supernatants from bone marrow cells stimulated for 24 h with 3P-RNA or R848.
(C) IFN-β in supernatants from DC preconditioned for 24 with poly(I:C) h and stimulated for 24 h with CpG, R848 and 3P-RNA.
****, p < 0.0001; “n.d.” cytokine levels below detection limit. Black bars vs. white bars. Student’s T-Test. Data are representative of at least 2 independent experiments.
3P-RNA p(I:C) + Lipo R848 none 0 200 400 600 800 IL -6 (p g /ml ) n.d. n.d. n.d. 3P-RNA R848 none 0 200 400 600 IL -6 (p g /ml ) n.d. n.d. 3P-RNA R848 none 0 100 200 300 400 IL -1 2 p 7 0 (p g /ml ) n.d. 3P-RNA CpG R848 none 0 200 400 600 800 IF N -α ( U/ m l) 3P-RNA R848 none 0 500 1000 1500 2000 2500 IL -1 2p40 ( p g/ m l) A J774
B Bone marrow cells
3P-RNA p(I:C) + Lipo) R848 none 0 500 1000 1500 IL -1 0 (p g /ml ) n.d. n.d. n.d. 3P-RNA R848 CpG none 0 500 1000 1500 IF N -β ( U /m l) no prestim. p(I:C) prestim. n.d. n.d. **** C
2
SUPPLEMENTAL FIGURE 2
(A) IL-6 and IFN-α in supernatants from TRIF or MDA-5-deficient DC preconditioned for 24 h with poly(I:C) and stimulated for 24 h with R848 and 3’-triphosphate RNA complexed with Lipofectamine (3P-RNA).
(B) IL-6 and IFN-α in supernatants from J774 cells preconditioned for 24 h with comnplexed poly(I:C) or LPS and stimulated for 24 h with R848 and 3’-triphosphate RNA. IL -6 (p g /ml ) R848 none 0 2000 4000 6000 8000 no precond. p(I:C) precond. * n.d. IL -6 (p g /ml ) R848 none 0 200 400 600 800 no precond. p(I:C) precond. ** n.d. n.d. 3P-RNA none 0 500 1000 1500 2000 2500 IF N -α ( U /m l) no precond. p(I:C) precond. **** n.d. n.d. IF N -α ( U /m l) 3P-RNA none 0 50 100 150 no precond. p(I:C) precond. **** MDA-5-/- DC TRIF-/- DC TLR pa thwa y R L R pa thwa y IL -6 (p g /ml ) R848 none 0 5000 10000 15000 20000 no precond. p(I:C) + Lipo precond.
** IF N -α ( U /m l) p(I:C) + Lipo none 0 500 1000 1500 2000 no precond. p(I:C) + Lipo precond.
n.d.
Preconditioning with MDA-5 and TLR4 ligands
IL -6 (p g /ml ) R848 none 0 500 1000 1500 no precond. LPS precond. *** n.d. IF N -α ( U /m l) p(I:C) + Lipo none 0 200 400 600 no precond. LPS precond. *** A B TLR pa thwa y R L R pa thwa y
*, p < 0.05; **, p < 0.01; ***, p< 0.001 and ****, p < 0.0001; “n.d.” cytokine levels below detection limit. Black bars vs. white bars. Student’s T-Test. Data are representative of at least 2 independent experiments (mean + s.e.m. of triplicates).
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SUPPLEMENTAL FIGURE 3
(A) IL-6 and IFN-α in supernatants from J774 cells preconditioned for 24 h with poly(I:C) and stimulated with R848 (left) or 3P-RNA (middle, right) for 24 h. In some conditions α-IFN-α, β or IFNaR antibodies were added in the preconditioning phase.
no AB no AB α-IFNaR 0 2000 4000 6000 IL -6 (p g /ml ) p(I:C) precond. **** no AB no AB α-IFNaR 0 400 800 1200 IF N - α (U /m l) p(I:C) precond. ** 0 4 8 12 16 20 24 0.0 0.5 1.0 1.5 2.0 2.5 duration of preconditioning (h) IL -6 ( fol d c h ange) *** no AB α-IFNaR 0.0 0.5 1.0 1.5 IFN -α ( fold c h ange) ) no prestim. p(I:C) precond. **** WT IFNaR -/-0 1 2 3 4 IL -6 ( fold c hange) no precond.. p(I:C) precond. *** WT IFNaR -/-0 1 2 3 4 IL -6 ( fold c hange) no precond.. p(I:C) precond. ** no AB α-IFNaR 0 1 2 3 4 IL -6 (re l. se cre ti o n ) no precond. p(I:C) precond. *** *** no AB no AB α- IFN-α α- IFN-β 0 200 400 600 IF N -α ( U /m l) p(I:C) precond. *** *** duration of prestimulation (h) IF N -α ( fol d c h ange) 0 4 8 12 16 20 24 0.0 0.5 1.0 1.5 2.0 *** RIG-I pathway TLR7 pathway
TLR7 pathway MDA-5 pathway
A
D
TLR7 pathway
TLR7 pathway RIG-I pathway
TLR9 pathway B
(B) IL-6 and IFN-α in supernatants from J774 preconditioned for different durations with recombinant IFN-β prior to stimulation for 24 h with R848 (left) or complexed poly(I:C) (right). Relative expression to the 0 h time-point is shown.
(C) IL-6 and IFN-α in supernatants from DCs stimulated from WT or IFNaR -/- 129/Sv mice preconditioned for 24 h with poly(I:C) and stimulated with R848 (left) or CpG (right) for 24 h. Data are expressed as fold change compared to the WT control. and are representative of 3 independent experiments (mean + s.e.m. of triplicates).
(D) IL-6 and IFN-α in supernatants from WT DCs preconditioned for 24 h with poly(I:C) and stimulated with R848 (left) or 3P-RNA (right) for 24 h. In some conditions α-IFNaR antibodies were added in the preconditioning phase. Data are expressed as fold change compared to non-antibody-treated, non-preconditioned control and are representative of 2 independent experiments (mean + s.e.m. of triplicates).
**, p < 0.01; ***, p< 0.001 and ****, p < 0.0001. (A,C,D): black bars vs. white bar or as indicated. Student’s T-Test (2-3 groups) or 1-way ANOVA, Dunnett’s Multiple Comparison Test (4 groups); (B): Zero h time-point vs. other conditions, 1-way ANOVA, Dunnett’s Multiple Comparison Test. Data are representative of 2-3 independent experiments (mean + s.e.m. of triplicates in A,C,D). Mean ± s.e.m. of 6 independent experiments are shown in (B).
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SUPPLEMENTAL FIGURE 4
Immunoblot analysis of p38 phosphorylation, TBK-1 and IRF-3 expression in lysates of J774 cells preconditioned for 8 and 24 h with poly(I:C) or IFN-β prior to stimulation for 60 and 120 min with complexed poly(I:C). Individual blots are depicted by rectangles. Some blots were sliced for clarity as indicated by dashed lines.
