Research ArticleIMMUNOLOGY

TRAF6-IRF5 kinetics, TRIF, and biophysical factors drive synergistic innate responses to particle-mediated MPLA-CpG co-presentation

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Science Advances  13 Jan 2021:
Vol. 7, no. 3, eabd4235
DOI: 10.1126/sciadv.abd4235
  • Fig. 1 Synergistic cytokine responses from BM-APCs induced by PLPs with MPLA and CpG depend on CpG adjuvant density.

    (A) Schematic of particle formulations for the co-delivery of MPLA and CpG. (B to F) Murine GM-CSF–differentiated murine BM-APCs (300,000 cells per well) were treated with formulations of varying size and CpG ligand density. IFN-β, IL-12p70, IL-6, TNF-α, and IL-10 in cell supernatants 24 hours after treatment. Each data point represents an independently treated well (n = 5). Center lines designate the mean value, and error bars represent SD. ***P < 0.001 and ****P < 0.0001; NS, not significant; one-way analysis of variance (ANOVA) with Tukey’s multiple comparison test.

  • Fig. 2 Knockdown of the adaptor protein TRIF ablates synergy from PLPs with MPLA and CpG in BM-APCs.

    (A) Schematic showing early signaling through the adaptor proteins MyD88 and TRIF following activation of TLR4 and TLR9. (B) IFN-β production from ΒΜDCs derived from WT, MyD88−/−, and TRIF−/− mice after PLP treatment. (C) IL-12p70 production from BM-APCs derived from WT, MyD88−/−, and TRIF−/− mice after PLP treatment. Each data point represents an independently treated well (n = 5 to 6). Center lines designate the mean value, and error bars represent SD. *P < 0.05 and ****P < 0.0001; NS, not significant; one-way ANOVA with Tukey’s multiple comparison test.

  • Fig. 3 IRF5, not IRF3 or IRF7, drives innate immune response triggered by MPLA and CpG in BM-APCs.

    (A) Diagram of TLR4 and TLR9 downstream signaling. (B) IFN-β production from BM-APCs derived from WT, IRF3−/−, IRF5−/−, and IRF7−/− mice. (C) IL-12p70 production from BM-APCs derived from WT, IRF3−/−, IRF5−/−, and IRF7−/− mice. We used the same WT BM-APCs in Fig. 1 for WT IL-12p70, IFN-β, TNF-α, IL-6, and IL-10 responses. Each data point represents an independently treated well (n = 5). Center lines designate the mean value, and error bars represent SD. ***P < 0.001 and ****P < 0.0001; NS, not significant; one-way ANOVA with Tukey’s multiple comparison test.

  • Fig. 4 Sustained IRF5 phosphorylation, higher IRF5 nuclear translocation, and elevated TRAF6 are responsible for synergistic IFN and IL-12p70 responses in BM-APCs.

    (A) Schematic showing TRAF6 and IRF5 signaling following TLR4 and TLR9 activation. (B) Levels of phosphorylated (Phos-tag) and total IRF5 [SDS–polyacrylamide gel electrophoresis (PAGE)] after 0.5, 1, 2, 4, 6, and 24 hours of BM-APCs treated with MPs with MPLA and/or CpG. GAPDH (glyceraldehyde-3-phosphate dehydrogenase) was used as a loading control, and treated IRF5−/− BM-APCs were used as negative controls. (C) Total IRF5 levels in the nuclear and cytoplasmic fractions at 1, 2, 4, 6, 12, and 24 hours of BM-APC treated with MPs with MPLA and/or CpG. Ratio of nuclear-to-cytoplasmic IRF5 after 1, 2, 4, 6, 12, and 24 hours of treatment, where a higher ratio indicates a higher rate of nuclear translocation. Ratios were performed by Bio-Rad Image Lab software. (D) Kinetics of IFN-β and IL-12p70 production by BM-APCs treated with PLPs. BM-APCs from this experiment were used for nuclear fractionation studies, and the results are shown in (C). (E) Levels of total TRAF6 (SDS-PAGE) after 0.5, 1, 2, 4, 6, and 24 hours of BM-APCs treated with MPs with MPLA and/or CpG. GAPDH was used as a loading control, and TRAF6 knocked down in BM-APCs was used as a negative control. On the right is the graphical representation of the GAPDH normalization of each blot. This was done with Bio-Rad Image Lab software.

  • Fig. 5 Schematic showing the mechanism of synergistic IFN-β and IL-12p70 cytokine response because of MPLA and CpG co-presentation on PLPs in BM-APCs.

Supplementary Materials

  • Supplementary Materials

    TRAF6-IRF5 kinetics, TRIF, and biophysical factors drive synergistic innate responses to particle-mediated MPLA-CpG co-presentation

    P. Pradhan, R. Toy, N. Jhita, A. Atalis, B. Pandey, A. Beach, E. L. Blanchard, S. G. Moore, D. A. Gaul, P. J. Santangelo, D. M. Shayakhmetov, K. Roy

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