Endosomal trafficking inhibitor EGA can control TLR7-mediated IFNα expression by human plasmacytoid dendritic cells

Abstract

Plasmacytoid dendritic cells (pDC) are the major producer of type 1 IFN in response to TLR7 agonists. Aberrant TLR7 activation and type 1 IFN expression by pDCs are linked to the pathogenesis of certain types of autoimmune diseases, including systemic lupus erythematosus (SLE). This study investigated the underlying mechanisms for TLR7-mediated cytokine expression by pDCs using a late endosome trafficking inhibitor, EGA (4-bromobenzaldehyde N-(2,6-dimethylphenyl) semicarbazone). We found that EGA treatment decreased IFNα expression by pDCs stimulated with imiquimod (R837), single-stranded RNA40, and influenza virus. EGA also decreased TNFα expression and secretion by R837-stimulated pDCs. Mechanistically, EGA treatment decreased phosphorylation of IKKα/β, STAT1, and p38, and prolonged degradation of IκBα. Furthermore, EGA treatment decreased the colocalization of 3F, a substituted adenine TLR7 agonist, with LAMP1⁺ compartments in pDCs. EGA was also capable of diminishing IFNα expression by SLE pDCs treated with R837 or live PR8/A/34 influenza viruses. Therefore, we concluded that trafficking of TLR7 agonists to LAMP1⁺ compartments is important for IFNα expression by pDCs. Data from this study support additional examinations of the potential benefits of EGA in treating type 1 IFN-associated inflammatory diseases in the future.

Keywords: EGA; TLR7; endosome trafficking; innate immunity; nucleic acid; plasmacytoid dendritic cells; proinflammatory cytokine; type 1 interferon.

Figure 1

EGA diminishes IFNα and TNFα secretion by R837-stimulated pDCs. FACS-sorted pDCs were pre-incubated for 30 mins with 20 μM EGA or vehicle. pDCs were then stimulated with 5 μg/mL R837 for 5 hours and stained for intracellular IFNα expression. Representative FACS plots (A). Summarized data of the frequency of intracellular IFNα⁺ pDCs (B). After overnight culture, the amount of IFNα in culture supernatants was measured by bead-based cytokine assays (C). Representative FACS plots of intracellular TNFα expression (D) and summarized data (E). The amount of TNFα in culture supernatants (F). Both IFNα (G) and TNFα (H) transcripts were measured by qRT-PCR. In (B–G, E–H), individual lines indicate data generated with pDCs from different subjects. Data analyzed by paired t-test. *p < 0.05, **p < 0.01, and n.s., not significant, for the comparison between groups.

Figure 2

EGA is more effective than YM201636 at controlling IFNα expression by R837-stimulated pDCs. FACS-sorted pDCs were pre-incubated for 30 mins with 20 μM EGA, 1 μM YM201636, or vehicle. pDCs were then stimulated with 5 μg/mL R837 for 5 hours and stained for intracellular IFNα expression. Representative FACS plots (A) and summarized data of the frequency of IFNα⁺ pDCs (B). After overnight culture, the amount of IFNα in the culture supernatants (C) was measured by bead-based cytokine assays. Representative FACS plots of the frequency of intracellular TNFα⁺ pDCs (D), summarized data of the frequency of intracellular TNFα⁺ pDCs (E), and the amount of TNFα secreted in the culture supernatants (F). In (B, C, E, F), individual lines indicate data generated with pDCs from different healthy subjects. Data analyzed by two-way ANOVA with Tukey multiple comparison test. *p <0.05, **p < 0.01, and ***p <0.001 for the comparison between groups.

Figure 3

EGA inhibits activation of IKKα/β, p38, and STAT1 but prolongs IκBα degradation. FACS-sorted pDCs were rested for 1 hour before treating them with 20 μM EGA, 1μM YM201636, or vehicle for 30 mins. Cells were then stimulated with 5 μg/mL R837 for the indicated time. Representative blot (A) with quantified changes in p-IKKα/β (B), IκBα (C), p-p65 (D), p-p38 (E), p-STAT1 (F), IRF7 (G), and ATF3 (H) from 3 independent experiments utilizing pDCs isolated from different donors. Inhibitor-mediated changes in protein data analyzed by two-way ANOVA with Tukey multiple comparison test. *p < 0.05, **p < 0.01, and ***p < 0.001 are for the comparison between groups.

Figure 4

EGA inhibits 3F-AF488 colocalization with LAMP1⁺ compartments in pDCs. (A) Reaction conditions for conjugation of 3F compound to Alexa Fluor 488. (B) HEK Blue hTLR7-expressing cells were stimulated with 10 μM 3F and 3F-AF488, and vehicle for 15 hours. SEAP expression was measured by optical density to assess NF-κB activity. Parental cell line (Null-1k) of hTLR7-expressing cells did not express secreted embryonic alkaline phosphatase (SEAP) in response to 3F stimulation. Representative data from experiment performed in triplicate assay. (C–E) pDCs were pre-incubated with 20 μM EGA, 1μM YM201636 or vehicle for 1 hour before addition of 20 μM 3F-AF488 for 5 hours. (C, D) Representative staining of endo-lysosomal markers and 3F-AF488 within pDCs. (E) Summarized colocalization data of 3F-AF488 with each endo-lysosomal marker in 30-34 different cells from 3 different experiments. Scale bar - 2μm. Data analyzed by one way ANOVA (B) or two-way ANOVA (E) with Tukey’s multiple comparison test. *p < 0.05, ****p <0.0001, and n.s., not significant for comparison between groups.

Figure 5

EGA reduces IFNα expression by SLE pDCs stimulated with R837 and PR8 influenza virus. PBMCs from SLE patients and healthy subjects were pre-incubated for 30 mins with 20 μM EGA or DMSO vehicle. Cells were then stimulated with 5 μg/mL R837 or 2 MOI PR8 influenza virus (A/PR8/34, H1N1) for 5 hours before staining them for intracellular IFNα and TNFα expression. Representative FACS plots for IFNα (A, left panels) and TNFα (B, left panels) expression by pDCs of healthy donors (HD) and SLE patients. Summarized data for IFNα (A, right panel) and TNFα (B, right panel). Individual lines indicate data generated with PBMCs from different subjects. Data analyzed by 2-way ANOVA with Tukey multiple comparison test. *p < 0.05, **p < 0.01, ***p < 0.001, ****p <0.0001, and n.s., not significant for comparison between groups.

Figure 6

EGA controls PR8 influenza virus-induced IFNα and R837-induced TNFα expression by mDC/monocyte population isolated from SLE patients. PBMCs from SLE patients and healthy subjects were pre-incubated for 30 mins with 20 μM EGA or vehicle. Cells were then stimulated with 5 μg/mL R837 or 2 MOI PR8 influenza virus (A/PR8/34, H1N1) for 5 hours before staining them for intracellular IFNα and TNFα expression. Representative FACS data for IFNα (A, left panels) and TNFα (B, left panels) expression by mDC/monocyte population from healthy donors (HD) and SLE patients. Summarized data for IFNα (A, right panel) and TNFα (B, right panel). Individual lines indicate data generated with PBMCs from different subjects. Data analyzed by 2-way ANOVA with Tukey multiple comparison test *p < 0.05, ***p < 0.001, and ****p <0.0001 and n.s., not significant for comparison between groups.