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. 2006 Apr 28;281(17):11678-84.
doi: 10.1074/jbc.M513286200. Epub 2006 Mar 3.

NFkappaB negatively regulates interferon-induced gene expression and anti-influenza activity

Lai Wei  1 , Matthew R Sandbulte, Paul G Thomas, Richard J Webby, Ramin Homayouni, Lawrence M Pfeffer
Affiliations

NFkappaB negatively regulates interferon-induced gene expression and anti-influenza activity

Lai Wei et al. J Biol Chem. .

Abstract

Interferons (IFNs) are antiviral cytokines that selectively regulate gene expression through several signaling pathways including nuclear factor kappaB(NFkappaB). To investigate the specific role of NFkappaB in IFN signaling, we performed gene expression profiling after IFN treatment of embryonic fibroblasts derived from normal mice or mice with targeted deletion of NFkappaB p50 and p65 genes. Interestingly, several antiviral and immunomodulatory genes were induced higher by IFN in NFkappaB knock-out cells. Chromatin immunoprecipitation experiments demonstrated that NFkappaB was basally bound to the promoters of these genes, while IFN treatment resulted in the recruitment of STAT1 and STAT2 to these promoters. However, in NFkappaB knock-out cells IFN induced STAT binding as well as the binding of the IFN regulatory factor-1 (IRF1) to the IFN-stimulated gene (ISG) promoters. IRF1 binding closely correlated with enhanced gene induction. Moreover, NFkappaB suppressed both antiviral and immunomodulatory actions of IFN against influenza virus. Our results identify a novel negative regulatory role of NFkappaB in IFN-induced gene expression and biological activities and suggest that modulating NFkappaB activity may provide a new avenue for enhancing the therapeutic effectiveness of IFN.

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Figures

Figure 1
Figure 1
Dose-dependent induction by IFN of Ifi47 (A), Tap1 (B) and Mx1 (C) expression. Real-time PCR was performed on cDNAs prepared from MEFs (WT, NFκB-KO and p50-KO) treated with IFNβ at varying concentrations for 5 hrs. Gene expression was normalized to actin expression in each sample. Data are shown as fold-induction relative to untreated fibroblasts, and are mean values ± the SEM (n=3).
Figure 2
Figure 2
Time course of IFN induced gene expression. (A) Real-time PCR was performed for Ifi47, Tap1, Mx1 and IRF1 using cDNAs prepared from MEFs treated with IFNβ at 1000 U/ml for varying times. Gene expression was normalized to actin expression in each sample. Data are shown as fold-induction relative to untreated fibroblasts, and are mean values +/- the SEM (n=3). (B) Immunoblotting was performed with indicated antibodies on whole cell or nuclear lysates prepared from MEFs treated with IFNβ (1000 U/ml) for the indicated times. Protein loading was evaluated by immunoblotting with anti-actin (cell) or -TFIIB (nuclear) antibodies. Similar results were obtained in at least two independent experiments.
Figure 3
Figure 3
Transcription factors binding profiles to NFκB-regulated ISG promoters in WT and NFκB-KO fibroblasts. ChIP assays were performed on extracts from control and IFN-treated MEFs using the indicated antibodies for precipitation and various primers that targeted specific regions in the Ifi47 (A), Tap1 (B), and Mx1 (C) promoters (see methods). Similar results were obtained in at least three independent experiments.
Figure 4
Figure 4
IFN-induced antiviral response to influenza infection in WT and NFκB-KO fibroblasts. To determine the ability of IFNβ to reduce virus titers in influenza-infected WT and NFκB-KO MEFs, fibroblasts were pre-incubated overnight with IFNβ, infected with influenza-PR8 virus, and at 24 hrs the virus yield was assayed by plaque formation (9). Viral titers in untreated WT and NFκB-KO MEFs were 9.5 ± 4.5 X 104 and 7.6 ± 2.2 X 104 pfu/ml, respectively.
Figure 5
Figure 5
IFN-induced immune response to influenza infection in WT and NFκB-KO fibroblasts. To determine the effect of IFN on the immune response to influenza virus, (A) the MHC class I levels (10), or (B) MHC class I antigen presentation (11) on control and IFN-treated MEFs was determined. The data represent the average of duplicate determinations from three independent experiments.
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