IκB kinase epsilon (IKK(epsilon)) regulates the balance between type I and type II interferon responses

Abstract

Virus infection induces the production of type I and type II interferons (IFN-I and IFN-II), cytokines that mediate the antiviral response. IFN-I (IFN-α and IFN-β) induces the assembly of IFN-stimulated gene factor 3 (ISGF3), a multimeric transcriptional activation complex composed of STAT1, STAT2, and IFN regulatory factor 9. IFN-II (IFN-γ) induces the homodimerization of STAT1 to form the gamma-activated factor (GAF) complex. ISGF3 and GAF bind specifically to unique regulatory DNA sequences located upstream of IFN-I- and IFN-II-inducible genes, respectively, and activate the expression of distinct sets of antiviral genes. The balance between type I and type II IFN pathways plays a critical role in orchestrating the innate and adaptive immune systems. Here, we show that the phosphorylation of STAT1 by IκB kinase epsilon (IKKε) inhibits STAT1 homodimerization, and thus assembly of GAF, but does not disrupt ISGF3 formation. Therefore, virus and/or IFN-I activation of IKKε suppresses GAF-dependent transcription and promotes ISGF3-dependent transcription. In the absence of IKKε, GAF-dependent transcription is enhanced at the expense of ISGF3-mediated transcription, rendering cells less resistant to infection. We conclude that IKKε plays a critical role in regulating the balance between the IFN-I and IFN-II signaling pathways.

Fig. 1.

IKKε knockout MEFs are susceptible to IAV infection due to an IFN-I–signaling defect. (A) Ikbke^+/+ and Ikbke^−/− MEFs were infected with A/Puerto Rico/8/34 at a multiplicity of infection of 1. Cells were harvested 0, 6, 12, and 24 h post infection (hpi). Recombinant type I IFNβ was added to the media at the beginning of the 24-h infection time point. Cell extracts were analyzed by Western blot for nucleocapsid (NP), hemaggluttin (HA1), and matrix (M1) protein expression. Actin was used as a loading control. (B) Western blot analysis of Ikbke^+/+ and Ikbke^−/− MEFs stimulated with IFNβ at the time points indicated. Blots depict protein levels of IFIT1, IFIT2, IRF1, and STAT1. Actin was used as a loading control. Experiments were independently repeated at least three times.

Fig. 2.

Loss of IKKε impacts IFN-I– and IFN-II–mediated transcription. qPCR from RNA derived from Ikbke^+/+ and Ikbke^−/− MEFs stimulated with IFNβ (A) or IFNγ (B) at the time points indicated. Genes analyzed include Ifit2, Mda5, Viperin, Stat1, Irf1, Irf8, and Icam1. All samples were normalized to Actin and standardized to unstimulated wild-type cells. Error bars represent SDs from triplicate measurements. Experiments were independently repeated at least three times.

Fig. 3.

ISGF3 binding decreases, whereas GAF binding increases in IKKε knockout MEFs. (A) EMSA with extracts derived from Ikbke^+/+ and Ikbke^−/− MEFs stimulated with IFNβ for the time points indicated. EMSA probes include IKKε-independent ISRE, IKKε-dependent ISRE, or IRF GAS element. (B) EMSA with extracts derived from Ikbke^+/+ and Ikbke^−/− MEFs stimulated with IFNβ or IFNγ for time points indicated and analyzed as in A. Unbound probe was used as loading control. Experiments were independently repeated at least three times.

Fig. 4.

IKKε phosphorylation disrupts the STAT1 homodimer, but not the STAT1:STAT2 heterodimer interface. (A) Space-filling diagram of the S708 (yellow) region and (B) hydrogen-bonding interactions of S708 in the STAT1:STAT1 homodimerization interface (light and dark blue). The diagram is adapted from the 3D structure published by Chen et al. (36) using PyMol. (C) 293T cells were transfected with HA STAT1 and FLAG STAT1 antiparallel (AP) mutants and GFP (mock transfected), IKKε WT, or IKKε K38A. Cells were unstimulated or stimulated with IFNγ. (D) 293T cells were transfected with HA STAT1 and FLAG STAT2 and GFP, IKKε WT, or IKKε K38A. Cells were unstimulated or stimulated with IFNβ. Coimmunoprecipitation experiments were performed using anti-HA matrix beads for the immunoprecipitation (IP) and immunoblotted (IB) for anti-FLAG tagged protein. Input samples were loaded as a control. Experiments were independently repeated at least three times.

Fig. 5.

Stat1α peaks near type-I and type-II ISGs are associated with different sequence motifs. (A) Motif frequencies in negative BGM (type-I) and positive BGM (type-II) ISG-associated peaks and control regions. For each peak associated with an ISG, a 100-bp region centered on the peak summit was extracted. As controls, each of these regions was dinucleotide shuffled (42), and also the 100 bp flanking each called peak was taken. The fraction of dinucleotide shuffled (dishuff) or flanking region (flank) controls, or real summits, containing at least one ISRE motif or GAS motif is shown. (B) Motifs discovered by unbiased search using BioProspector in summit regions (100 bp) associated with negative and positive BGM ISGs.