Atg9a controls dsDNA-driven dynamic translocation of STING and the innate immune response

Abstract

Microbial nucleic acids are critical for the induction of innate immune responses, a host defense mechanism against infection by microbes. Recent studies have indicated that double-stranded DNA (dsDNA) induces potent innate immune responses via the induction of type I IFN (IFN) and IFN-inducible genes. However, the regulatory mechanisms underlying dsDNA-triggered signaling are not fully understood. Here we show that the translocation and assembly of the essential signal transducers, stimulator of IFN genes (STING) and TANK-binding kinase 1 (TBK1), are required for dsDNA-triggered innate immune responses. After sensing dsDNA, STING moves from the endoplasmic reticulum (ER) to the Golgi apparatus and finally reaches the cytoplasmic punctate structures to assemble with TBK1. The addition of an ER-retention signal to the C terminus of STING dampens its ability to induce antiviral responses. We also show that STING co-localizes with the autophagy proteins, microtubule-associated protein 1 light chain 3 (LC3) and autophagy-related gene 9a (Atg9a), after dsDNA stimulation. The loss of Atg9a, but not that of another autophagy-related gene (Atg7), greatly enhances the assembly of STING and TBK1 by dsDNA, leading to aberrant activation of the innate immune response. Hence Atg9a functions as a regulator of innate immunity following dsDNA stimulation as well as an essential autophagy protein. These results demonstrate that dynamic membrane traffic mediates the sequential translocation and assembly of STING, both of which are essential processes required for maximal activation of the innate immune response triggered by dsDNA.

Fig. 1.

Double-stranded DNA triggers the assembly of STING and TBK1. Wild-type MEFs stably expressing STING-Myc were transfected with poly (dA-dT) (1 μg/mL) for the indicated periods. The localization of STING and the indicated proteins was observed by confocal laser scanning microscopy. (Scale bars, 20 μm.)

Fig. 2.

The translocation of STING from ER facilitates to induce antiviral response. (A) Luciferase assay of lysates from 293 cells transfected with various amounts of the indicated STING expression vectors along with pISRE-Luc and pRL-TK. ISRE, IFN-stimulation responsive element. TK, thymidine kinase. Firefly luciferase activity was normalized to renilla luciferase activity to measure ISRE-dependent transcriptional activity. The results shown are means ± SD. (n = 3). (B) Anti-HSV1 activity in culture supernatants from 293 cells transfected with the indicated expression vectors for 24 h. HSV1 production [in 50% tissue culture infectious dose (TCID50)] was measured 24 h after infection of Vero cells either untreated or treated with the indicated culture supernatants.

Fig. 3.

STING colocalizes with Atg9a and assembles at unique membrane-bound compartments after dsDNA stimulation. (A and B) Wild-type MEFs stably expressing STING-Myc were transfected with poly (dA-dT) (1 μg/mL) for the indicated periods. The localization of STING and the indicated proteins was observed by confocal laser scanning microscopy. (Scale bars, 20 μm.) (C) Wild-type primary MEFs stably expressing STING-GFP were transfected with poly (dA-dT) for 8 h and then fixed. The cells were subjected to fluorescence-coupled electron microscopy. Yellow arrow indicates the STING -positive membrane-bound compartments.

Fig. 4.

Atg9a controls the assembly of STING and TBK1 after dsDNA stimulation. Atg9a^+/+ and Atg9a^Δ/Δ primary MEFs stably expressing STING-Myc were transfected with poly (dA-dT) (1 μg/mL) for the indicated periods. The cells were fixed, immunostained with anti-Myc and anti-TBK1 antibodies and then observed by confocal laser scanning microscopy. (Scale bars, 20 μm.)

Fig. 5.

Loss of Atg9a enhances dsDNA-induced innate immune response. (A) Immunoblots of lysates from Atg9a^+/+ and Atg9a^Δ/Δ primary MEFs transfected with poly (dA-dT) for the indicated periods. The membranes were probed with the indicated antibodies. (B) Quantitative-RT-PCR of the indicated RNAs from Atg9a^+/+ and Atg9a^Δ/Δ primary MEFs transfected with poly (dA-dT) for 8 h. The results shown are means ± SD. (n = 3). (C) Production of IFN-β by Atg9a^+/+ and Atg9a^Δ/Δ primary MEFs transfected with poly (dA-dT) for 24 h. The results shown are means ± SD. (n = 3).