Fig 1: 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.
Fig 2: Schematic model of Irf8R294C -mediated impairment in immune response. Upon NDV infection, IRF8WT induces type I IFN promoter and transcription initiates. Type I IFN further results in production of downstream ISGs and pDC activation. Thus, together, type I IFN and ISGs restrict viral replication and mount successful immune response. Whereas, IRF8R294C fails to induce type I IFN promoter, and transcription does not take place. So, the absence of type I IFN leads to impaired pDC activation and abrogation of production of ISGs concomitant with increased NDV burden. Thus, IRF8R294C results in dysregulated immune response.
Fig 3: Impaired type I IFN production in IRF8R294C pDCs leads to impairment in induction of ISGs. Transcript analysis of Irf7, Oas3, Oasl2, Oas1g, Oas1a, Ifit3, and Oas2 by qRT-PCR in FLDC cultures from IRF8WT and IRF8R294C mice stimulated with CpG (A) or infected with NDV (B) at indicated time points. Data (A, B) are representative of three independent experiments with error bar representing ± SEM.
Fig 4: 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 5: 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.
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