STING is a direct innate immune sensor of cyclic di-GMP - PubMed (original) (raw)

STING is a direct innate immune sensor of cyclic di-GMP

Dara L Burdette et al. Nature. 2011.

Abstract

The innate immune system detects infection by using germline-encoded receptors that are specific for conserved microbial molecules. The recognition of microbial ligands leads to the production of cytokines, such as type I interferons (IFNs), that are essential for successful pathogen elimination. Cytosolic detection of pathogen-derived DNA is one major mechanism of inducing IFN production, and this process requires signalling through TANK binding kinase 1 (TBK1) and its downstream transcription factor, IFN-regulatory factor 3 (IRF3). In addition, a transmembrane protein called STING (stimulator of IFN genes; also known as MITA, ERIS, MPYS and TMEM173) functions as an essential signalling adaptor, linking the cytosolic detection of DNA to the TBK1-IRF3 signalling axis. Recently, unique nucleic acids called cyclic dinucleotides, which function as conserved signalling molecules in bacteria, have also been shown to induce a STING-dependent type I IFN response. However, a mammalian sensor of cyclic dinucleotides has not been identified. Here we report evidence that STING itself is an innate immune sensor of cyclic dinucleotides. We demonstrate that STING binds directly to radiolabelled cyclic diguanylate monophosphate (c-di-GMP), and we show that unlabelled cyclic dinucleotides, but not other nucleotides or nucleic acids, compete with c-di-GMP for binding to STING. Furthermore, we identify mutations in STING that selectively affect the response to cyclic dinucleotides without affecting the response to DNA. Thus, STING seems to function as a direct sensor of cyclic dinucleotides, in addition to its established role as a signalling adaptor in the IFN response to cytosolic DNA. Cyclic dinucleotides have shown promise as novel vaccine adjuvants and immunotherapeutics, and our results provide insight into the mechanism by which cyclic dinucleotides are sensed by the innate immune system.

PubMed Disclaimer

Conflict of interest statement

The authors declare no competing financial interests.

Figures

Figure 1

Figure 1. STING is sufficient to restore responsiveness to cyclic dinucleotides

a, Immortalized Myd88 −/−Trif −/− macrophages were transduced with retrovirus expressing RocR and stimulated for 6h. SeV, Sendai Virus; TMEV, Theiler’s Virus. IFN induction was measured by qRT-PCR and normalized to ribosomal protein 17 (Rps17). (b–d) HEK293T cells were transfected as indicated along with an IFN-luciferase reporter and luciferase activity was measured 6h following stimulation. VV 70-mer is a stimulatory dsDNA oligo derived from Vaccinia virus. ***, P < 0.001 e, Myd88 −/−Trif −/_−_macrophages were stimulated for 6h and IFN induction was measured as in a. Data are mean ± s.d. (n = 3) and are representative of at least three independent experiments.

Figure 2

Figure 2. STING binds cyclic dinucleotides

a, HEK293T cells were transfected as indicated and cell lysates were subjected to an in-vitro UV-crosslinking assay with c-di-GMP32. Samples were separated by SDS-PAGE and visualized by autoradiography or western blotting. b, HEK293T cells were transfected as in a and anti-HA immunoprecipitates were subjected to the c-di-GMP32 binding assay or stained with Colloidal Blue. c and d, HEK293T cells were transfected as in a and cell lysates were UV-crosslinked to c-di-GMP32 or GTP32 in the presence of cold competing nucleotides in 10-fold serial dilutions beginning at 1 mM, except for guanosine (0.1 mM), VV 70mer (500 μg/mL), poly(dAT:dTA) (50 μg/mL) and poly(I:C) (50 μg/mL). Arrow, STING; Asterisk, nonspecific bands. e, 1μg His6-mSTING 138–378 was separated by SDS-PAGE and stained with coomassie. f, His6-mSTING was analyzed as in c. g, C-di-GMP binding to purified His6-mSTING (10μM) was measured by equilibrium dialysis. Data are representative of three independent experiments.

Figure 3

Figure 3. Mutational analysis of STING

a, HEK293T cells were transfected as indicated along with an IFN-luciferase reporter and luciferase activity was measured 6h following stimulation. b, HEK293T cells were transfected as in a except the lysates were subjected to the c-di-GMP32 binding assay as in Fig. 2a. c, Organization of STING based on the membrane topology prediction programs SOSUI, TMHMM, HMMTOP and TMPRED. Strongly predicted transmembrane domains are boxed; weakly predicted transmembrane domains have dashed boxes. Colored residues indicate mutant classes (see text): Class I (red), Class II (purple), Class III (green), Class IV (blue) and Class V (yellow). Bracketed mutations were made in combination. Data are representative of at least three independent experiments. Data are mean ± s.d. (n = 3).

Figure 4

Figure 4. The IFN response to DNA and c-di-GMP can be uncoupled

a, HEK293T cells were transfected and IFN reporter activity was measured 6h following stimulation. b, HEK293T cells were transfected as in a except the lysates were subjected to the c-di-GMP32 binding assay as in Fig. 2a. c, Bone marrow-derived macrophages from _Sting_-deficient goldenticket mice were transduced with the indicated constructs. IFN induction by transfected cyclic-di-GMP and VV 70-mer dsDNA was measured by qRT-PCR and normalized to ribosomal protein 17 (Rps17). ***, P < 0.001. ns, not significant, P = 0.1205) Data are representative of at least three independent experiments. Data are mean± s.d. (n = 3).

Similar articles

Cited by

References

    1. Ishii KJ, et al. A Toll-like receptor-independent antiviral response induced by double-stranded B-form DNA. Nat Immunol. 2006;7:40–48. - PubMed
    1. Stetson DB, Medzhitov R. Recognition of cytosolic DNA activates an IRF3-dependent innate immune response. Immunity. 2006;24:93–103. - PubMed
    1. Ishikawa H, Barber GN. STING is an endoplasmic reticulum adaptor that facilitates innate immune signalling. Nature. 2008;455:674–678. - PMC - PubMed
    1. Ishikawa H, Ma Z, Barber GN. STING regulates intracellular DNA-mediated, type I interferon-dependent innate immunity. Nature. 2009;461:788–792. - PMC - PubMed
    1. Jin L, et al. MPYS, a novel membrane tetraspanner, is associated with major histocompatibility complex class II and mediates transduction of apoptotic signals. Mol Cell Biol. 2008;28 - PMC - PubMed

Publication types

MeSH terms

Substances

Grants and funding

LinkOut - more resources