Figure 3.
NRF2 inhibits IFN-I response in pDCs by activating the PPP. (A–D) Purified pDCs from HDs (n = 3) were cultured in media alone or with the NRF2 activator (KI696) for 1 h, followed by the TLR9 agonist (C274 at 0.075 μM) for 5 h and analyzed by RNA-seq. (A) PCA plot of RNA-seq analysis. (B) All differentially expressed genes identified by RNA-seq were analyzed for pathway analysis using gene set enrichment analysis. NES, normalized enrichment score. (C) IPA identified pathways that were significantly activated or inhibited by the NRF2 activator (KI696) in TLR9-activated pDCs, and upregulated and downregulated pathways are indicated in red and blue, respectively. (D) Heatmaps showing the genes that are involved in the PPP. (E) Purified pDCs from HDs (n = 4; three independent experiments) were cultured at a stiffness of 0 or 50 kPa for 1 h, followed by incubation with the TLR9 agonist (C274 at 0.075 μM) for 5 h. TKT and G6PD gene expressions were analyzed by Q-PCR. (F–J) Purified pDCs from HDs (n = 3) were cultured in media alone or with the NRF2 activator (KI696) for 1 h, followed by incubation with the TLR9 agonist (C274-0.075 μM) for 5 h. Cells were collected for CUT&RUN assay. Analysis of NRF2 CUT and RUN data obtained using pDCs from three independent donors and treated as indicated above. (F) PCA plot of NRF2 data for media, TLR9-L, KI696, and TLR9-L + KI696 treatment of primary human pDCs. (G) UpSet plot showing the overlap between differentially upregulated peaks induced by TLR9-L and TLR9-L + KI696 compared to resting media-treated pDCs. (H) Heatmap of the NRF2 normalized signal density surrounding peaks identified in Fig. 3 G and plotted under the indicated conditions. Results are presented in reads per kilobase per million mapped reads (RPKM) values within a range of ±3.0 kb around peak centers. (I) De novo motif analysis results using HOMER of NRF2 binding peaks that are shared by TLR9-L, and TLR9-L + KI696. (J) A representative IGV gene track is shown, illustrating treatment-induced NRF2 binding at the HMOX1, G6PD, and TKT loci. (K) Purified pDCs from HDs (n = 5; three independent experiments) were cultured in media alone or with the NRF2 activator (KI696) alone or with sodium pyruvate (10 mM) for 1 h, followed by incubation with the TLR9 agonist for 5 h. IFNA2 gene expression was analyzed by Q-PCR. (L) Purified pDCs from HDs (n = 4–5; three independent experiments) were transfected with CRISPR-Cas9 complex targeting for G6PD and TKT on exon 2 and exon 3 respectively using Lipofectamine CRISPRMAX reagent and cultured with IL3 (20 ng/ml) for 72 h. Cultured pDCs were treated alone or with the NRF2 activator (KI696) for 1 h, followed by incubation with the TLR9 agonist (C274-0.3 μM) for 5 h. IFNA2 gene expression was analyzed by Q-PCR. Individual donors are indicated; all results are represented as mean ± SEM; and statistical significance was evaluated using Mann-Whitney U test or one-way ANOVA, Tukey’s multiple comparisons test. *P < 0.05; ***P < 0.001. Refer to the image caption for details.

NRF2 inhibits IFN-I response in pDCs by activating the PPP . (A–D) Purified pDCs from HDs (n = 3) were cultured in media alone or with the NRF2 activator (KI696) for 1 h, followed by the TLR9 agonist (C274 at 0.075 μM) for 5 h and analyzed by RNA-seq. (A) PCA plot of RNA-seq analysis. (B) All differentially expressed genes identified by RNA-seq were analyzed for pathway analysis using gene set enrichment analysis. NES, normalized enrichment score. (C) IPA identified pathways that were significantly activated or inhibited by the NRF2 activator (KI696) in TLR9-activated pDCs, and upregulated and downregulated pathways are indicated in red and blue, respectively. (D) Heatmaps showing the genes that are involved in the PPP. (E) Purified pDCs from HDs (n = 4; three independent experiments) were cultured at a stiffness of 0 or 50 kPa for 1 h, followed by incubation with the TLR9 agonist (C274 at 0.075 μM) for 5 h. TKT and G6PD gene expressions were analyzed by Q-PCR. (F–J) Purified pDCs from HDs (n = 3) were cultured in media alone or with the NRF2 activator (KI696) for 1 h, followed by incubation with the TLR9 agonist (C274-0.075 μM) for 5 h. Cells were collected for CUT&RUN assay. Analysis of NRF2 CUT and RUN data obtained using pDCs from three independent donors and treated as indicated above. (F) PCA plot of NRF2 data for media, TLR9-L, KI696, and TLR9-L + KI696 treatment of primary human pDCs. (G) UpSet plot showing the overlap between differentially upregulated peaks induced by TLR9-L and TLR9-L + KI696 compared to resting media-treated pDCs. (H) Heatmap of the NRF2 normalized signal density surrounding peaks identified in Fig. 3 G and plotted under the indicated conditions. Results are presented in reads per kilobase per million mapped reads (RPKM) values within a range of ±3.0 kb around peak centers. (I) De novo motif analysis results using HOMER of NRF2 binding peaks that are shared by TLR9-L, and TLR9-L + KI696. (J) A representative IGV gene track is shown, illustrating treatment-induced NRF2 binding at the HMOX1, G6PD, and TKT loci. (K) Purified pDCs from HDs (n = 5; three independent experiments) were cultured in media alone or with the NRF2 activator (KI696) alone or with sodium pyruvate (10 mM) for 1 h, followed by incubation with the TLR9 agonist for 5 h. IFNA2 gene expression was analyzed by Q-PCR. (L) Purified pDCs from HDs (n = 4–5; three independent experiments) were transfected with CRISPR-Cas9 complex targeting for G6PD and TKT on exon 2 and exon 3 respectively using Lipofectamine CRISPRMAX reagent and cultured with IL3 (20 ng/ml) for 72 h. Cultured pDCs were treated alone or with the NRF2 activator (KI696) for 1 h, followed by incubation with the TLR9 agonist (C274-0.3 μM) for 5 h. IFNA2 gene expression was analyzed by Q-PCR. Individual donors are indicated; all results are represented as mean ± SEM; and statistical significance was evaluated using Mann-Whitney U test or one-way ANOVA, Tukey’s multiple comparisons test. *P < 0.05; ***P < 0.001.

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