Abstract
Stimulator of interferon genes (STING) is an endoplasmic reticulum-resident membrane protein that mediates cytosolic pathogen DNA-induced innate immunity and inflammatory responses in host defenses. STING is activated by cyclic di-nucleotides and is then translocated to the Golgi apparatus, an event that triggers STING assembly with the downstream enzyme TANK-binding kinase 1 (TBK1). This assembly leads to the phosphorylation of the transcription factor interferon regulatory factor 3 (IRF3), which in turn induces expression of type-I interferon (IFN) and chemokine genes. STING also mediates inflammatory responses independently of IRF3, but these molecular pathways are largely unexplored. Here, we analyzed the RAW264.7 macrophage secretome to comprehensively identify proinflammatory factors released into the extracellular medium upon STING activation. In total, we identified 1299 proteins in macrophage culture supernatants, of which 23 were significantly increased after STING activation. These proteins included IRF3-dependent cytokines, as well as previously unknown targets of STING, such as the immune semaphorin SEMA4D/CD100, which possesses proinflammatory cytokine-like activities. Unlike for canonical cytokines, the expression of the SEMA4D gene was not up-regulated. Instead, upon STING activation, membrane-bound SEMA4D was cleaved into a soluble form, suggesting the presence of a post-translational shedding machinery. Importantly, the SEMA4D shedding was blocked by TMI-1, an inhibitor of the sheddase ADAM metallopeptidase domain 17 (ADAM17) but not by the TBK1 inhibitor BX795. These results suggest that STING activates ADAM17 and that this activation produces soluble proinflammatory SEMA4D independently of the TBK1/IRF3-mediated transcriptional pathway.
Highlights
Stimulator of interferon genes (STING) is an endoplasmic reticulum–resident membrane protein that mediates cytosolic pathogen DNA–induced innate immunity and inflammatory responses in host defenses
We identified novel STING-dependent signaling in which a cell surface protein, SEMA4D, is cleaved to produce its soluble ectodomain
The increased concentration of soluble SEMA4D in serum or plasma is associated with numerous inflammatory conditions in humans and mice [26], and the soluble SEMA4D has been described as a stimulator of both innate and acquired immunity [26]
Summary
To determine the inflammatory factors released from STING-activated cells, we prepared culture supernatants from Raw264.7 murine macrophage cells treated with or without DMXAA, a cell-permeable synthetic STING agonist. We identified 1299 proteins, of which 23 proteins were significantly increased (q Ͻ 0.05, -fold increase Ͼ 4) by DMXAA stimulation (Fig. 1B, Table 1, and Table S1) These 23 significantly increased proteins included IRF3-dependent cytokines such as IFN- and CXCL10, and the most increased protein was TNF␣ (about 150-fold increase). The protein levels of SEMA4D, LRPAP1, and Fam3C in culture supernatants were increased about 3– 6-fold by DMXAA (Fig. 1C and Table S2). These observations prompted us to speculate that STING activates transcription and post-translational machinery to release SEMA4D and TNF␣ To confirm this possibility, the cells were treated with CHX to inhibit de novo protein synthesis and stimulated with DMXAA.
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