Fig 1: Nrf2 is an endogenous suppressor of IFN-ß in inflammatory macrophages(A) IFN-ß, IL-6, and TNF protein levels in LPS-stimulated Nrf2-KO and Keap1-KD compared to WT cells (n = 3 biological replicates).(B) Ifnb and Ifit2 mRNA levels in LPS-stimulated Nrf2-KO and Keap1-KD compared to WT cells (n = 5-6 biological replicates).(A-B) Data are mean ± SEM p value determined by unpaired t test, corrected for multiple comparisons by Holm-Sidak test.(C) IFN-ß in Poly(I:C) stimulated Nrf2-KO and Keap1-KD compared to WT cells (n = 3 biological replicates). Data are mean ± SEM p value determined by two-tailed unpaired t test.(D) Ifnb and Ifit2 mRNA levels in WT, Nrf2-KO, and Keap1-KD cells that had been pre-treated for 1 h with the Nrf2 activator TBE-31 (30 nM) and stimulated with LPS (100 ng/mL) for a further 4 h (n = 3 biological replicates).(E) Ifnb and Ifit2 mRNA levels in WT, Nrf2-KO, and Keap1-KD cells that had been pre-treated for 24 h with the Nrf2 activator TBE-31 (20 nM) and stimulated with LPS (100 ng/mL) for a further 4 h (n = 2-3 biological replicates).(D-E) Data are mean ± SEM p value determined by one-way ANOVA, corrected for multiple comparisons by Tukey statistical test.(F) Nqo1 mRNA levels in WT, Nrf2-KO, and Keap1-KD cells that had been pre-treated for 24 h with the Nrf2 activator TBE-31 (20 nM) (n = 2-3 biological replicates). Data are mean ± SEM p value determined by a two-tailed unpaired t test. p <0.05*; p <0.01**; p <0.001***.
Fig 2: IRF8R294C pDCs show abrogation of type I IFN production in vitro. (A) RNA was isolated from IRF8WT and IRF8R294C FLDCs and analyzed by qRT-PCR to measure Ifnb expression. FLDC cultures from IRF8WT and IRF8R294C mice were stimulated with CpG (1 µg/ml) for 8 h, and level of Ifnb (B) and Ifna (C) transcript expression was checked by qRT-PCR. FLDC cultures from IRF8WT and IRF8R294C mice infected with NDV for 8 h were checked for expression of Ifnb (D), Ifna (E) transcripts, and NDV abundance (F) by qRT-PCR. FLDC culture supernatants from IRF8WT and IRF8R294C mice stimulated with CpG or infected with NDV for 24 h were examined for expression of IFN-ß (G), IFN-a1 (H) cytokine by ELISA. FLDC cultures from IRF8WT and IRF8R294C mice stimulated with CpG (1 µg/ml) for indicated time points were analyzed for Ifna (I) and Ifnb (J) transcripts by qRT-PCR. FLDC cultures from IRF8WT and IRF8R294C mice infected with NDV for indicated time points were analyzed for Ifna (K) and Ifnb (L) transcripts by qRT-PCR. Data (A–F) are representative of three independent experiments with error bar representing + standard error of mean (SEM). Data (G, H) are mean of three independent experiments with error bar representing + standard error of mean (SEM). Data (I–L) are representative of three independent experiments with error bar representing ± standard error of mean (SEM). ND, not detected; *p < 0.05, **p < 0.01, ***p < 0.001; p value obtained from Student’s t test.
Fig 3: Defect of type I IFN production by IRF8R294C pDCs is not due to increased cell death. FLDC cultures from IRF8WT and IRF8R294C mice were kept unstimulated or stimulated with CpG and stained for different surface markers followed by 7-AAD staining. Cells with negative staining for 7-AAD were considered as live cells. Flow cytometric analysis (A) and quantitation (B) of live CD11c+ B220+ SiglecH+ pDCs from IRF8WT and IRF8R294C FLDC cultures after 6 h time point. Flow cytometric analysis (C) and quantitation (D) of live CD11c+ B220+ SiglecH+ pDCs from IRF8WT and IRF8R294C FLDC cultures after 24 h time point. IRF8WT activates expression of luciferase reporter construct driven by Ifnb (E) and Ifna6 (F) promoters, while IRF8R294C was unable to induce the expression of promoters. Data (A, C) are representative of three independent experiments. Data (B, D, E, F) are mean of three independent experiments with error bar representing + SEM and ***p < 0.001, ns, non-significant. p value obtained from Student’s t test.
Fig 4: Impairment of type I IFN production in IRF8R294C mice upon NDV infection. (A) Schematic representation of the workflow for in vivo NDV infection. Transcript analysis of Ifna (B), Ifnb (C), and abundance of NDV (D) in splenocytes from uninfected or NDV-infected IRF8WT and IRF8R294C mice by qRT-PCR. IFN-a1 (E) and IFN-ß (F) concentration in sera from uninfected or NDV-infected IRF8WT and IRF8R294C mice were quantitated by ELISA. Data (B–D) shown are mean value with error bar representing + SEM (n= 6 mice per group). Data (E, F) shown are mean value with error bar representing + SEM (n= 3 mice per group) and *p < 0.05, **p < 0.01, ***p < 0.001; p value obtained from Student’s t test.
Supplier Page from Abcam for Mouse IFN beta ELISA Kit