Skip to main page content
Access keys NCBI Homepage MyNCBI Homepage Main Content Main Navigation
. 2017 Feb 10;8:14391.
doi: 10.1038/ncomms14391.

IFI16 Is Required for DNA Sensing in Human Macrophages by Promoting Production and Function of cGAMP

Affiliations
Free PMC article

IFI16 Is Required for DNA Sensing in Human Macrophages by Promoting Production and Function of cGAMP

K L Jønsson et al. Nat Commun. .
Free PMC article

Abstract

Innate immune activation by macrophages is an essential part of host defence against infection. Cytosolic recognition of microbial DNA in macrophages leads to induction of interferons and cytokines through activation of cyclic GMP-AMP synthase (cGAS) and stimulator of interferon genes (STING). Other host factors, including interferon-gamma inducible factor 16 (IFI16), have been proposed to contribute to immune activation by DNA. However, their relation to the cGAS-STING pathway is not clear. Here, we show that IFI16 functions in the cGAS-STING pathway on two distinct levels. Depletion of IFI16 in macrophages impairs cGAMP production on DNA stimulation, whereas overexpression of IFI16 amplifies the function of cGAS. Furthermore, IFI16 is vital for the downstream signalling stimulated by cGAMP, facilitating recruitment and activation of TANK-binding kinase 1 in STING complex. Collectively, our results suggest that IFI16 is essential for efficient sensing and signalling upon DNA challenge in macrophages to promote interferons and antiviral responses.

Conflict of interest statement

The authors declare no competing financial interests.

Figures

Figure 1
Figure 1. The immune response to HIV and HSV infection in human macrophages is regulated by IFI16.
(a) IFI16 expression was measured by western blotting in four primary human macrophage donors treated with scrambled (Sc.) and IFI16-specific (IFI16) siRNA. (b,c) ISG54 expression (b) and IFN-β expression (c) was measured in the four donors portrayed in (a) challenged with either Vpx particles alone or HIVvpx+ for 18 h. Data represent mRNA expression of each gene normalized to mRNA expression of RNaseP from two biological replicates of each donor. (d) IFI16 expression measured by WB in four primary human macrophages treated with scramble or IFI16-specific siRNA at the time of challenge with HIV-Bal infection (left panel). Production of replication competent HIV particles was measured 6 days p.i. using the HIV permissive TzmBL Luciferase reporter cell line (right panel). (e,f) Type I interferon expression was evaluated in control, IFI16 KO, cGAS KO and STING KO THP-1 cells challenged with HSV1 (e) or hCMV (f) 18 h after infection using a MOI of 3. (g) Type I interferon expression was evaluated in control and IFI16 KO cells at 2, 4 and 8 h after HSV1 infection using a MOI of 10. Data in (eg) represent the mean±s.d. of biological triplicates, representative of two independent experiments. Unpaired t-test corrected for multiple comparisons using Holm–Sidak was been performed to evaluate the significance. *P<0.05; **P<0.01.
Figure 2
Figure 2. Cytosolic DNA sensing and efficient innate signalling is dependent on IFI16.
(a) Control and IFI16 CRISPR KO THP-1 cells were transfected with dsDNA at various concentrations and interferon induction measured after 6 h. (b,c) Control and IFI16 KO cells were transfected with dsDNA (4 μg ml−1) at indicated time-points (b) or poly (I:C) (1 μg ml−1 or 5 μg ml−1) for 20 h (c), hereafter supernatants were evaluated for type I interferon expression. (d) Whole cell lysates from control or IFI16 KO cells stimulated with dsDNA (4 μg ml−1) at indicated time-points were subjected to immunoblotting using antibodies against STING, pIRF3, pTBK1, total TBK, total IRF3 and vinculin (VCL) as loading control. (e) Control or IFI16 KO cells were transfected with dsDNA (4 μg ml−1) for two and four hours. The cells were fixed and stained with anti-IFI16 (Green) and anti-STING (Red) specific antibodies. DNA was visualized with DAPI (blue). Data represent mean±s.d. of biological triplicates, representative of three independent experiments. Unpaired t-test corrected for multiple comparisons using Holm–Sidak was been performed to evaluate the significance. *P<0.05; **P<0.01; ***P<0.001.
Figure 3
Figure 3. STING dimerization and phosphorylation is dependent on IFI16.
(a,b) Control and IFI16 KO THP-1 cells were stimulated with dsDNA (4 μg ml−1) at indicated time-points and whole cell lysates were subjected to immunoblotting of STING dimerization by semi-native gel electrophoresis. Vinculin (VCL) was used as loading control. (b) The quantification of band intensity of STINGDimer versus STINGMonomor was done using ImageJ software of three independent experimental setups. (c,d) Control, cGAS KO and IFI16 KO cells were stimulated with dsDNA (4 μg ml−1) at indicated time-points and whole cell lysates were subjected to both semi-native gel electrophoresis and standard SDS–page. Membranes were probed with antibodies against STING, p-TBK1 and VCL (c) or phospho-specific STING Ser366 and Histone3 as loading control (d). Data presented in (a,c) are representative of at least three independent experiments, whereas data in (d) is representative of two independent experiments.
Figure 4
Figure 4. ISG expression profile in IFI16-deficient cells.
(a) Control and IFI16 KO cells were stimulated with dsDNA (4 μg ml−1) for 6 h before extracting total RNA and analysing differentially expressed genes on ProtonIon with Partek Gene Specific Analysis Algoritm. (b) Total gene expression (number of reads normalized to total reads) are presented for the 6 red-marked genes: IFI44L, VIPERIN, IFNB1, MX1, APOBEC3F and GBP5. The box represents interquartile range, with the line in the middle representing the median while the whiskers symbolize 90–10% range.
Figure 5
Figure 5. Recruitment of TBK1 to STING is dependent on IFI16 interactions.
(a) Schematic illustration of the workflow of co-immunoprecipitation experiments. Cleared cell lysates (CCL) of THP-1 cells stimulated with dsDNA (4 μg ml−1) for 2 and 4 h were subjected to over-night co-immunoprecipitation with antibodies indicated in each panel. Lysates from control cells were co-IP with STING (lane 1–3) or IFI16 (lane 4–6). Input and elutes were analysed by gel electrophoresis followed by immunoblotting (IB) with the indicated antibodies. (b) STING co-IP samples from primary human MDMs after IB with the indicated antibodies. (c) STING co-IP samples from control (lane 1–3) and IFI16 KO (lane 4–6) THP-1 cells after IB with the indicated antibodies. (d) IFI16 co-IP samples from STING KO THP-1 cells after IB with the indicated antibodies. Each blot is representative of three independent experiments. (e) Control or IFI16 KO cells were stimulate with dsDNA (4 μg ml−1) for 2 h, fixed and stained for DAPI (blue), anti-IFI16 (green) or anti-IRF3 (red) and subjected to confocal imaging at × 63 oil lens. (f) Quantification of IRF3 localization of at least 50 individual cells treated as described in e.
Figure 6
Figure 6. cGAMP production is regulated by IFI16.
(a) External calibration curve of spiked (2′3′-3′5′)-cGAMP into cell extract before column purification were used to quantify cGAMP production in stimulated cells. The calibration curve was linear up to a concentration of at least 400 nM with an R2 of 0.991. The chromatogram demonstrates the peak detected using synthetic cGAMP. (b) LC-MS/MS chromatograms of whole cell lysates from control and IFI16 KO THP-1 cells stimulated with dsDNA for 2, 4 or 8 h. (c) Quantitative LC–MS/MS analysis of control and IFI16 KO THP-1 of three individual single clones. (d) Immunoblotting of HEK29T with or without stable transduction of human IFI16 (CE, cytoplasmic extract; ME, membrane extract; NE, nuclear extract; PE, pellet extract). (e) Quantitative LC–MS/MS analysis of HEK293T with or without stable transduction of human IFI16 24 h after transfection with increasing doses of cGAS expressing plasmid. (f) Immunoblotting of HEK293T with or without stable transduction of human STING. (g) HEK293TSTING cells were transfected with cGAS expressing plasmid (25 ng well−1) and increasing doses of IFI16 expressing plasmid (0, 250, 500, 750 and 1,000 ng well−1). STING activation was evaluated 24 h later by measuring expression of an IFN-β promoter Firefly gene normalized to a beta-actin promotor Renilla gene. (h) Diagram of IFI16 domains and the two different IFI16-mutants used to transient express IFI16 protein in HEK293T stable expressing human STING. An eGFP expressing plasmid was used as negative control. Transfection efficiencies were evaluated by measuring eGFP or BFP by Flow cytometry. (i) HEK293TSTING cells were transfected with cGAS expressing plasmid (25 ng well−1) and increasing doses of plasmids expressing wt, Pyrin or Hin IFI16 mutant. (j) HEK293TSTING cells were transfected with cGAS expressing plasmid (25 ng well−1) and increasing doses of plasmids expressing Pyrin containing proteins; MNDA, IFIX or IFI16. Data in (c,e,g,i,j) represent mean±s.d. of biological triplicates from three independent experimental setups. Unpaired t-test corrected for multiple comparisons using Holm–Sidak was performed to evaluate the significance. For data in (i) one-way ANOVA was performed to evaluate significance. *P<0.05; **P<0.01, ***P<0.001.
Figure 7
Figure 7. IFI16 regulates cGAMP-mediated STING activation.
(a) Control, IFI16, cGAS, STING KO THP-1 cells or (b) MDMs with IFI16 siRNA knockdown, were infused with cGAMP (50 nM) at indicated time-points and subsequently evaluated for type I interferon secretion. (c) STING dimerization analysis by semi-native western blotting. Upper lane represents an overexposure of the dimer STING band. Total STING was run on a separate SDS–Page gel. (d) Control and IFI16 KO cells were infused with cGAMP (50 nM) for 2 h, fixed and stained for DAPI (blue), IFI16 (green) and IRF3 (red). (e) IRF3 translocation from cytoplasm to nuclear saturation were quantified by counting >50 separate images of control or IFI16 KO cells 2 h post cGAMP infusion. (f) Subcellular fractions of control and IFI16 KO cells stimulated with 50 nM cGAMP for 1 h were immunoblotted for phosphorylated IRF3 and total IRF3 in cytosolic (cyto) and nuclear (nucl) fractions. Data in (a,b) represent mean±s.d. of biological triplicates from (a) three independent experimental setups or (b) one donor; (cf) data is representative of one of three independent experiments.
Figure 8
Figure 8. IFI16 regulates STING activation through its PYRIN domain.
(a) Control and TBK1 KO or (b) Control and IFI16 KO THP-1 cells were infused with cGAMP (50 nM) for 30 min, 1, 4 and 8 h and whole cell lysates was used to evaluate STING dimerization (upper panel) and specific STING phosphorylation at Ser366 (lower panel). Black arrows represent the phosphorylated form of the STING dimer. (c) HEK293TSTING-IFI16 expressing cells were infused with cGAMP (range from 50 to 250 nM) for 16 h and the degree of STING activation was evaluated by measuring expression of an IFN-β promoter Firefly gene normalized to a beta-actin promotor Renilla gene. (d) HEK293T- cGAS expressing cells were co-cultured with HEK293TSTING that had been transfected with eGFP or one of the three IFI16 variants. Twenty-four hours after culturing cGAMP transfer and STING activation was evaluated by measuring expression of IFN-β promoter Firefly gene normalized to beta-actin promotor Renilla gene. Data represent mean±s.d. of biological triplicates, representative of three independent experiments. Unpaired t-test corrected for multiple comparisons using Holm–Sidak was performed to evaluate the significance. *P<0.05; **P<0.01.

Similar articles

See all similar articles

Cited by 52 articles

See all "Cited by" articles

References

    1. Paludan S. R. Activation and regulation of DNA-driven immune responses. Microb. Mol. Biol. Rev. 79, 225–241 (2015). - PMC - PubMed
    1. Unterholzner L. The interferon response to intracellular DNA: why so many receptors? Immunobiology 218, 1312–1321 (2013). - PubMed
    1. Jakobsen M. R. & Paludan S. R. IFI16: at the interphase between innate DNA sensing and genome regulation. Cytokine Growth Factor Rev. 25, 649–655 (2014). - PubMed
    1. Brazda V., Coufal J., Liao J. C. & Arrowsmith C. H. Preferential binding of IFI16 protein to cruciform structure and superhelical DNA. Biochem. Biophys. Res. Commun. 422, 716–720 (2012). - PubMed
    1. Jakobsen M. R. et al. . IFI16 senses DNA forms of the lentiviral replication cycle and controls HIV-1 replication. Proc. Natl Acad. Sci. USA 110, E4571–E4580 (2013). - PMC - PubMed

Publication types

MeSH terms

LinkOut - more resources

Feedback