STING regulates intracellular DNA-mediated, type I interferon-dependent innate immunity

Abstract

The innate immune system is critical for the early detection of invading pathogens and for initiating cellular host defence countermeasures, which include the production of type I interferon (IFN). However, little is known about how the innate immune system is galvanized to respond to DNA-based microbes. Here we show that STING (stimulator of interferon genes) is critical for the induction of IFN by non-CpG intracellular DNA species produced by various DNA pathogens after infection. Murine embryonic fibroblasts, as well as antigen presenting cells such as macrophages and dendritic cells (exposed to intracellular B-form DNA, the DNA virus herpes simplex virus 1 (HSV-1) or bacteria Listeria monocytogenes), were found to require STING to initiate effective IFN production. Accordingly, Sting-knockout mice were susceptible to lethal infection after exposure to HSV-1. The importance of STING in facilitating DNA-mediated innate immune responses was further evident because cytotoxic T-cell responses induced by plasmid DNA vaccination were reduced in Sting-deficient animals. In the presence of intracellular DNA, STING relocalized with TANK-binding kinase 1 (TBK1) from the endoplasmic reticulum to perinuclear vesicles containing the exocyst component Sec5 (also known as EXOC2). Collectively, our studies indicate that STING is essential for host defence against DNA pathogens such as HSV-1 and facilitates the adjuvant activity of DNA-based vaccines.

Figure 1. STING is essential for intracellular DNA-mediated type I IFN production

a, MEFs were transfected with 1 μg ml⁻¹ of DNA ligands (with Lipofectamine 2000) for 16 h, and IFNβ or IL6 were measured. b, MEFs were transfected with ISD for 4 h and Ifnb or Ifna2 mRNA levels were measured. c, MEFs treated as in b were stained with an antibody for IRF3 translocation. Original magnification, ×40. d, Bone-marrow-derived macrophages were transfected with poly(dAT:dAT), poly(I:C) or ISD, or infected with HSV-1 (multiplicity of infection (m.o.i.) 10) or Listeria (m.o.i. 10) for 16 h, and IFNβ was measured. e, Macrophages were infected with HSV-1 for 16 h and IL1β was measured. f, GM-colony stimulating factor (CSF)-induced dendritic cells (GM-DCs) were treated as in d, and IFNβ or IFNα was measured after 16 h. g, FLT3-stimulated dendritic cells were treated as in f (exogenous CpG oligodeoxynucleotides (ODN) (1 μg ml⁻¹) were also used). *P < 0.05, Student's t-test. Error bars indicate s.d. ND, not determined.

Figure 2. STING is required for effective in vivo host defence

a, Sting-deficient animals (Sting^−/−) or littermate controls (Sting^+/+) (n = 7; approximately 8-weeks-of-age) were infected with HSV-1 (1 × 10⁷ i.v.) and survival was monitored. b, Sting^−/− or control mice were infected with HSV-1 as in a and brains were retrieved after 5 days for HSV-1 plaque assays. p.f.u., plaque-forming units. c, d, Serum from animals (n = 3) infected with HSV-1 (1 × 10⁷ i.v.) was analysed for IFNβ (c) or IFNα (b) production after 6 h. e, f, Serum from animals infected as in c was analysed for RANTES (e) and IL6 (f) production. g, Sting^−/− or control mice (n = 6) were infected with VSV (5 × 10⁷ i.v.) and survival was monitored. h, i, Mice (n = 3) were treated as in g and IFNβ (h) or IFNα (i) was measured after 6 h. j, Increasing amounts of YFV NS4B were co-transfected into 293T cells with human STING or the amino terminus of RIG-I (ΔRIG-I, residues 1–284) and transfected IFNβ promoter-driven luciferase (IFNβ-Luc) was measured after 36 h. k, Immortalized MEFs were transfected with YFV NS4B for 24 h, infected with VSVΔM⁴ (m.o.i. 1) for 16 h, and IFNβ was measured. l, 293 cells were transfected with NS4B–HA for 36 h and after immunoprecipitation (IP) with anti-haemagglutinin antibody, were analysed by western blot (WB) using anti-STING serum. *P < 0.05, Student's t-test. Error bars indicate s.d.

Figure 3. STING is required for effective DNA-mediated adaptive immune responses

a, Sting^−/− or control (Sting^+/+) mice (n = 5; approximately 8-weeks-of-age) were immunized twice (100 μg i.m.) by electroporation with a DNA vaccine encoding ovalbumin. Serum was measured for anti-OVA IgG. b, c, Mice were treated as in a and spleen CD8⁺ IFNγ⁺ cells were measured by fluorescence-activated cell sorting (FACS; b), and anti-OVA-specific IFNγ production was measured by ELISA after stimulation of splenocytes using SIINFEKL peptide (c). d, Sting^−/− mice or controls (n = 4; approximately 8-weeks-of-age) were infected with vaccinia expressing ovalbumin (VV-OVA; 5 × 10⁶ i.v.) and spleen anti-OVA-specific IFNγ production was measured by ELISA. *P < 0.05, Student's t-test. Error bars indicate s.d. All experiments were repeated twice.

Figure 4. STING translocates from the ER to Sec5-containing vesicles

a, Sting^−/− MEFs, stably reconstituted with haemagglutinin-tagged mouse STING (mSTING–HA) were stained using haemagglutinin (green) and a calreticulin (red) antibody. b, STING–HA MEFs were stained for STING–HA (green), calreticulin (blue) or Mitotracker (red) and three-dimensional reconstruction images were taken. c, Immunoblot analysis of fractionation experiments of uninfected or HSV-1-infected (m.o.i. 10; 4 h) MEFs. Endogenous STING was detected using an anti-STING antibody. Calreticulin detects ER, Sigma1R detects MAM, and COXIV detects mitochondria. d, Haemagglutinin (green) or calreticulin (red) staining of mSTING–HA MEFs after treatment with transfected ISD (1 μg ml⁻¹), transfected ssDNA (1 μg ml⁻¹) or HSV-1 infection as in c. e, mSTING–HA MEFs were transfected with or without ISD and cells were stained with haemagglutinin (green), calreticulin (blue) and a TBK1 (red) antibody. f, mSTING–HA MEFs were transfected as in e and stained with haemagglutinin (green) and a TFR (red) antibody. g, mSTING–HA MEFs were transfected as in e and stained with haemagglutinin (green) and a Sec5 antibody (red). h, i, MEFs were treated with RNAi to Trapb, Sting or Sec5 for 72 h and transfected with ISD. IFNβ mRNA and protein were measured at 4 and 16 h, respectively. *P < 0.05, Student's t-test. Error bars indicate s.d. Scale bars, 10 μm.