IFI16 is an innate immune sensor for intracellular DNA

Abstract

The detection of intracellular microbial DNA is critical to appropriate innate immune responses; however, knowledge of how such DNA is sensed is limited. Here we identify IFI16, a PYHIN protein, as an intracellular DNA sensor that mediates the induction of interferon-β (IFN-β). IFI16 directly associated with IFN-β-inducing viral DNA motifs. STING, a critical mediator of IFN-β responses to DNA, was recruited to IFI16 after DNA stimulation. Lowering the expression of IFI16 or its mouse ortholog p204 by RNA-mediated interference inhibited gene induction and activation of the transcription factors IRF3 and NF-κB induced by DNA and herpes simplex virus type 1 (HSV-1). IFI16 (p204) is the first PYHIN protein to our knowledge shown to be involved in IFN-β induction. Thus, the PYHIN proteins IFI16 and AIM2 form a new family of innate DNA sensors we call 'AIM2-like receptors' (ALRs).

Figure 1

Induction of IFNβ by a VACV DNA motif. a, HEK293T cells were transfected with nucleic acids (0.5 and 5 μg/ml) for 16 h and IFNβ promoter activity was measured by reporter gene assay. b, PMA-treated THP-1 cells were transfected with 1 μg/ml poly(dA-dT) or with 200 ng/ml DNA isolated from VACV, calf thymus or Listeria for 6 h and IFNβ mRNA was measured. c, d THP1 (c) or immortalized MEF (d) cells were transfected with 1 μg/ml (c) or 5 μg/ml (d) nucleic acids for 6 h and IFNβ mRNA was measured. e, HEK293T cells were transfected with 5 μg/ml nucleic acids for 16 h, and IFNβ promoter activity was measured. f, g, THP-1 (f) or RAW264.7 (g) cells were transfected with different lengths (b.p.) of 1 μg/ml VACV 70mer-derived DNA, GC-rich 70mer (70(GC)) or interferon stimulatory DNA (ISD) for 6 h and IFNβ mRNA was measured. Error bars indicate s.d.

Figure 2

Induction of IFNβ by a VACV DNA motif is independent of known DNA sensing pathways. a-f, h-j, Immortalised BMDMs or MEFs derived from mice lacking signalling components as indicated were transfected with 5 μg/ml VACV 70mer (a, c, e, h-j) or poly(dA-dT) (f) or stimulated with LPS (b) or poly(I:C) (d) for 6 h and IFNβ mRNA measured. Values are expressed as % of stimulation observed in cells from wild type mice. Data is from three independent experiments performed in triplicate and error bars represent s.e.m. g, Immortalised BMDMs were transfected with nucleic acid for 6h and IFNβ mRNA measured (left panel). 100 ng of the RNA extracted from BMDMs was transfected into HEK293T cells for 16 h, and IFNβ promoter activity measured (right panel). Data from one experiment of three is shown where error bars represent s.d.

Figure 3

IFI16 binds to immune stimulatory viral DNA. a, Schematic representation of human PYHINs. Aa, amino acids. b, Cytoplasmic extracts from PMA-treated THP-1 cells were incubated with biotinylated ss or dsVACV 70mer immobilised on streptavidin beads. Precipitated proteins were immunoblotted with an IFI16-specific antibody as indicated. c, Left panel: THP-1 cells were transfected with 1 μg/ml dsVACV 70mer or poly(I:C) in the absence or presence of 1 μg/ml ssVACV 70mer and IFNβ mRNA was measured after 6 h. Right panel: HEK293T cells were transfected with 50 ng/ml poly(dA-dT) alone, or together with 50 ng/ml ss or dsVACV 70mer and IFNβ promoter activity measured after 16 h. Data from one experiment of three is shown where error bars represent s.d. d, PMA/IFN α-treated THP-1 cells grown on coverslips were transfected with 2.5 μg/ml VACV 70mer or poly(I:C) for 1 h. Cells were fixed and stained with anti-IFI16 antibody (red). DNA and poly(I:C) were visualised with DAPI (blue). e, FITC-labelled HSV-1 60mer was transfected into PMA-treated THP-1 cells for 3 h. Cells were fixed and stained for IFI16 (red). DAPI-stained DNA is shown in blue (upper panel) and the HSV 60mer is shown in green (lower panel). Scale bar: 10 μm. f, AlphaScreen assessment of IFI16 HIN domains binding dsVACV 70mer. Left panel, 30 nM DNA with increasing concentration of HIN domains or GB1 expression tag. Right panel, 30 nM protein domain with increasing concentrations of biotin-70mer.

Figure 4

Role for STING in IFI16-mediated IFNβ induction. a, Cytoplasmic extracts from PMA-treated THP-1 cells were incubated with biotinylated ss or dsVACV 70mer immobilised on streptavidin beads. Precipitated proteins were immunoblotted as indicated. b, PMA/IFNα pre-treated THP1 cells were transfected with 1 μg/ml VACV 70mer for 4 h. Resulting lysates were incubated with anti-STING antibody and immuno-precipitated proteins were immunoblotted as indicated. c, Myc-tagged human (hu) or mouse (mur) STING were overexpressed in HEK293 cells, immobilized on sepharose beads, and incubated with lysates from DNA-treated THP1 cells. Co-immunoprecipitated IFI16 was detected by immunoblotting. Ab, antibody heavy chain. d, e, BMDMs lacking STING were transfected with either VACV 70mer , HSV 60mer or infected with HSV-1 or Sendai virus for 18 h as indicated and IFNβ protein release was measured by ELISA. f, Immortalised BMDMs from ASC-null mice were transfected with DNA as indicated for 18 h and IFNβ protein secretion was measured by ELISA. Data from one experiment of at least 2 (performed in triplicate) is shown where error bars represent s.d. (d-f).

Figure 5

IFI16/p204 is required for DNA-mediated gene induction. a, Left panel, IFI16 mRNA was measured in THP-1, PMA-treated THP-1, or HEK293 cells. Right panel, THP-1 or PMA-treated THP-1 cells were transfected with VACV 70mer for 6 h and IFNβ mRNA measured. b, c, siRNA-treated THP-1 cells were transfected with 1 μg/ml VACV 70mer for 6 h, before detection of IFI16 protein expression by immunoblotting (b) or measurement of IFNβ mRNA (c). d, Domain organization of human IFI16 compared to murine members of the PYHIN family, drawn to scale. Boxes represent conserved domains. aa, amino acids. Of the murine proteins, p204 (*) is most similar to human IFI16, in terms of domain structure and aa identity. e, f, siRNA-treated RAW264.7 cells were transfected with 1 μg/ml VACV 70mer for 6 h and p204 protein expression was analysed by immunoblotting (e) or p204, IFNβ, CCL5 and TNF mRNA was measured (f). g, siRNA-treated MEF cells were transfected with 1 μg/ml VACV 70mer for 6 h and p204, IFNβ, CCL5 and TNF mRNA was measured. h, siRNA-treated RAW264.7 cells were treated with HSV 60mer DNA for 6 h before measurement of IFNβ mRNA. i, BMDMs were electroporated with siRNA prior to transfection with oligomers for 18 h, and IFNβ protein release was measured. Data from one experiment of 2-3 (performed in triplicate) is shown (a, c, f-i) where error bars represent s.d. *P < 0.05 compared with control siRNA.

Figure 6

p204 is required for VACV 70mer DNA-stimulated transcription factor activation. a, siRNA-treated RAW264.7 cells grown on glass coverslips were mock-transfected or transfected with 2.5 μg/ml VACV 70mer or poly(I:C) for 6 h, fixed and stained for NF-κB p65 (red) and IRF3 (green). Nuclei were visualised by DAPI (blue). b, Cells treated as in (a) were qualitatively examined to assess staining of either p65 (left panel) or IRF3 (right panel) in the nucleus. Cells showing nuclear staining were counted and expressed as a percentage of total number of cells. At least 200 cells were counted per sample. Shown is a representative of four independent experiments.

Figure 7

IFI16/p204 is required for the innate immune response to HSV-1. a, BMDMs were infected with HSV-1 (MOI=10) for 6 h and IFNβ mRNA was measured. b, RAW264.7 cells were pre-treated with ML60812 for 2 h prior to infection with HSV-1 (MOI=10), or transfection with poly(dA-dT) for 6 h. c, d, siRNA-treated RAW264.7 cells were infected with HSV-1 or Sendai virus for 6 h and IFNβ (c, d), CXCL10 (c), IL-6 (c) and TNF (c) mRNA was measured. e, siRNA-treated RAW264.7 cells were infected with HSV1 (MOI 10) or Sendai virus for 20 h and IFNβ protein expression determined by ELISA. f, siRNA-treated RAW264.7 cells grown on glass coverslips were mock-infected or infected with HSV-1 or Sendai virus for 6 h, fixed and stained for NF-κB p65 (red) and IRF3 (green). Nuclei were visualised by DAPI (blue). g, Cells treated as in (f) were qualitatively examined to assess staining of either p65 (left panel) or IRF3 (right panel) in the nucleus. Cells showing nuclear staining were counted and expressed as a percentage of total number of cells. At least 200 cells were counted per sample. Shown is a representative of three independent experiments. h, RAW264.7 cells transfected with HSV 60mer (2 μg/ml) were infected with HSV-1 (MOI 1) and culture supernatants were harvested at the indicated time points post infection for viral quantification by plaque assay. Data from one experiment of 2-3 (performed in triplicate) is shown (a-e, h) where error bars represent s.d.*P < 0.001 compared with control siRNA.