cGAS-STING signalling is induced by detection of foreign or mislocalised host double-stranded (ds)DNA within the cytosol. STING acts as the major signalling hub, where it controls production of type I interferons and inflammatory cytokines. Basally, STING resides on the ER membrane. Following activation STING traffics to the Golgi to initiate downstream signalling and subsequently to endolysosomal compartments for degradation and termination of signalling. While STING is known to be degraded within lysosomes, the mechanisms controlling its delivery remain poorly defined.Here we utilised a proteomics-based approach to assess phosphorylation changes in primary murine macrophages following STING activation. This identified numerous phosphorylation events in proteins involved in intracellular and vesicular transport. We utilised high-temporal microscopy to track STING vesicular transport in live macrophages. We subsequently identified that the endosomal complexes required for transport (ESCRT) pathway detects ubiquitinated STING on vesicles, which facilitates the degradation of STING in murine macrophages. Disruption of ESCRT functionality greatly enhanced STING signalling and cytokine production, thus characterising a mechanism controlling effective termination of STING signalling.

TANK-binding kinase 1 (TBK1) is a key signalling component that drives the production of type-I interferons (IFNs), which have essential antiviral activities including against SARS-CoV-2. TBK1 and its homolog IκB kinase-ε (IKKε) can also induce the production of pro-inflammatory factors that contribute to pathogen clearance. While initially protective, delayed engagement of type-I IFN is associated with lethal hyper-inflammation seen in severe COVID-19 patients. The contribution of TBK1/IKKε signalling to this response is unknown. We have discovered that the small molecule idronoxil inhibits both IRF3 and NF-κB-driven inflammation by disrupting the formation of TBK1/IKKε signalling complexes following STING activation. Leveraging this unique activity, we show that therapeutic administration of idronoxil suppresses cellular and molecular lung inflammation in K18-hACE2 mice challenged with SARS-CoV-2, resulting in reduced pro-inflammatory cytokine production and decreased airway fibrosis in experimental COVID-19. Our results indicate a critical role for TBK1/IKKε signalling in SARS-CoV-2 hyper-inflammation and identify a novel therapeutic intervention to limit disease severity in COVID-19 patients.

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