Disruption of endosomal trafficking with EGA alters TLR9 cytokine response in human plasmacytoid dendritic cells

Abstract

Plasmacytoid dendritic cells (pDCs) exhibit bifurcated cytokine responses to TLR9 agonists, an IRF7-mediated type 1 IFN response or a pro-inflammatory cytokine response via the activation of NF-κB. This bifurcated response has been hypothesized to result from either distinct signaling endosomes or endo-lysosomal trafficking delay of TLR9 agonists allowing for autocrine signaling to affect outcomes. Utilizing the late endosome trafficking inhibitor, EGA, we assessed the bifurcated cytokine responses of pDCs to TLR9 stimulation. EGA treatment of pDCs diminished both IFNα and pro-inflammatory cytokine expression induced by CpG DNAs (D- and K-type), CpG-DNAs complexed with DOTAP, and genomic DNAs complexed with LL37. Mechanistically, EGA suppressed phosphorylation of IKKα/β, STAT1, Akt, and p38, and decreased colocalization of CpG oligodeoxynucleotides with LAMP⁺ endo-lysosomes. EGA also diminished type 1 IFN expression by pDCs from systemic lupus erythematosus patients. Therefore, our findings help understand mechanisms for the bifurcated cytokine responses by pDCs and support future examination of the potential benefit of EGA in treating type 1 IFN-associated inflammatory diseases in the future.

Keywords: EGA; endosomal trafficking; nucleic acid; plasmacytoid dendritic cells; pro-inflammatory cytokine; toll-like receptor 9; type 1 interferon.

Figure 1

EGA diminishes cytokine expression in pDCs stimulated with CpG-ODNs and influenza virus. (A–D) pDCs pre-incubated with 20 μM EGA or vehicle, stimulated with 2 MOI PR8 influenza virus (A/PR8, H1N1) or CpG-ODN2006 for 5 hours or CpG-ODN2216 overnight and stained for IFNα (A, B) and TNFα (C, D). Representative flow cytometry plots (A, C) and compiled data set (B, D) from 4-5 different subjects. (E) Cytokines in supernatants of pDCs pre-incubated with 20 μM EGA or vehicle and stimulated with 2 MOI PR8 influenza virus (A/PR8, H1N1) or TLR9 agonists overnight. Compiled data from 4-5 different subjects. Data analyzed by two-tailed paired t-test. *P< 0.05, **P< 0.01, ns, not significant, for comparison between groups.

Figure 2

Kinetics of EGA-mediated suppression of cytokine expression in pDCs stimulated with CpG-ODNs. (A, B) pDCs pre-incubated with indicated concentrations of EGA before stimulation with CpG-ODN2216. Representative flow cytometry plots (A) and compiled data of fold change relative to vehicle treated cells (B) after overnight stimulation from 4 different subjects. (C) pDCs stimulated with CpG-ODN2216 overnight with 20 μM EGA or vehicle added at time points post-CpG-ODN2216 stimulation. Fold changes in the supernatant cytokine concentration after overnight stimulation based on averaging concentration from all time points in vehicle treated cells as reference point. Data from 4 different subjects. Data analyzed by (B) 1-way ANOVA followed by Dunnett’s multiple comparison or (C) 2-way ANOVA followed by Sidak’s multiple comparison test. *P< 0.05, **P< 0.01, ***P< 0.001, ****P<0.0001, ns, not significant, for comparison between groups.

Figure 3

EGA affects cytokine production in CpG-ODN/DOTAP- and genomic DNA/LL37- stimulated pDCs. (A–D) pDCs pre-incubated with 20 μM EGA or vehicle, stimulated with free CpG-ODN or complexed to DOTAP for 5 hours and stained for IFNα and TNFα. Representative flow cytometry plots from one experiment (A, C) and compiled data (B–D) from 4 different subjects. (E, F) Blood pDCs pre-incubated with 20 μM EGA or vehicle, stimulated with genomic DNA and LL37 overnight, and stained for IFNα (E) and TNFα (F). Representative flow cytometry plots and compiled data from 4 different subjects. Data analyzed by two-tailed paired t-test. *P< 0.05, **P< 0.01, for comparison between groups.

Figure 4

EGA and PIKfyve inhibitor, YM201636, can reduce IFNα and TNFα expression by pDCs treated with CpG-ODN2216. (A–D) pDCs pre-incubated with 20 μM EGA, 1 μM YM201636, or vehicle, stimulated with CpG-ODN2216 overnight, and stained for IFNα and TNFα. Representative flow cytometry plots and compiled data for IFNα (A, B) and TNFα (C, D) from 5 different subjects. Data analyzed by 2-way ANOVA followed by Tukey multiple comparison test. *P< 0.05, ***P< 0.001, ****P<0.0001, for comparison between groups.

Figure 5

EGA and YM201636 differentially inhibit activation of kinases involved in endosomal trafficking and CpG-ODN-mediated cytokine expression. (A–I) pDCs pre-incubated with 20 μM EGA, 1 μM YM201636 or vehicle and stimulated with 1 μM CpG-ODN2216 for indicated time. Representative blot (A) with summarized quantified data changes in kinases (B–I). Representative of 3 independent experiments with kinase kinetics analyzed with vehicle-treated group by one-way ANOVA with Dunnett’s multiple comparison test. Inhibitor data analyzed by two-way ANOVA with Tukey multiple comparison test. *P< 0.05, **P< 0.01, ***P< 0.001, ****P<0.0001, for comparison between groups.

Figure 6

EGA and YM201636 treated pDCs have decreased colocalization of CpG-ODN2216-FITC with late endosome/lysosome markers. (A–C) Enriched pDCs pre-incubated with 20 μM EGA, 1μM YM201636 or vehicle for 1 hour before addition of 1 μM CpG-ODN2216-FITC for 2 hours. Representative staining of endo-lysosomal markers and CpG-ODN within pDCs (A, B) and summarized colocalization data (C) of 38 cells from 3 experiments. Scale bar 2μm. Data analyzed in (C) by 2-way ANOVA followed by Tukey multiple comparison test.*P< 0.05, **P< 0.01, ***P < 0.001, ****P<0.0001, for comparison between groups.

Figure 7

EGA affects TLR7/9 agonist responses in SLE pDCs. (A–C) PBMCs from SLE patients pre-incubated with 20 μM EGA, 1 μM YM201636, or vehicle, stimulated with CpG-ODN2006/DOTAP or 2 MOI PR8 influenza virus (A/PR8, H1N1) for 5 hours. Representative flow cytometry gating for pDCs (A) and IFNα and TNFα expression (B) and compiled data (C) from 7 different subjects. Data analyzed in (C) by 2-way ANOVA followed by Tukey multiple comparison test.*P< 0.05, **P< 0.01, ****P<0.0001, for comparison between groups.