Global transcriptional changes in response to cGAMP depend on STING in human THP-1 cells

Cytosolic detection of foreign or misplaced self-DNA through the cGAS-STING pathway leads to cGAS-mediated synthesis of the second messenger molecule cGAMP. The immune signaling molecule STING binds cGAMP directly, and thus functions as a cGAMP sensor. Binding of cGAMP induces the activation of STING with the subsequent induction of anti-viral and proinflammatory genes. Currently, it is not known whether STING is the only cellular sensor of cGAMP, and it has not been reported whether stimulation with cGAMP can induce gene transcription independently of STING. Here we analyzed the global cGAMP-induced transcriptional gene program in STING-deficient human monocyte-derived THP-1 cells. We found that the global cGAMP-induced transcriptional response was dependent on STING and failed to identify groups of genes that were affected by cGAMP in the absence of STING. Our data therefore strongly indicate that STING is the sole receptor for cGAMP in human macrophages.

The innate immune system is the first line of defense against invading microorganisms and cancer cells. Incoming pathogens are sensed through a restricted number of receptors known as Pattern Recognition Receptors (PRRs), which recognize distinct evolutionarily conserved microbial structures termed Pathogen-Associated Molecular Patterns (PAMPs). Innate virus detection is highly dependent on intracellular sensors of viral nucleic acids, which include members of the Toll-like receptor family (TLRs), which detect DNA and RNA in endosomes, and receptors that recognize non-self RNA and DNA in the cytosol.¹

Recently, cyclic GMP-AMP synthase (cGAS) was identified as the most critical sensor of cytosolic DNA.^2–4 Direct binding of DNA to cGAS induces a structural rearrangement that switches its enzymatic activity on and leads to the formation of a small second messenger, cGAMP (cyclic GMP-AMP).⁵ cGAMP binds to the endoplasmic reticulum (ER)-anchored protein STING (stimulator of interferon genes), which subsequently interacts with TBK1, and together, they translocate from the ER to the Golgi. TBK1 then phosphorylates STING, which is a necessary step for the recruitment and activation of the transcription factor Interferon Regulatory Factor 3 (IRF3). IRF3 then relocates to the nucleus and initiates the transcription of type I interferon (IFN) and other antiviral genes.^5–8

Cyclic diguanylate monophosphate (c-di-GMP), cyclic diadenylate monophosphate (c-di-AMP) and cGAMP are all second messengers produced by bacteria or human cells to regulate complex biological processes. Recently, the cyclic bacterial cousin of cGAMP, c-di-AMP, was shown to directly interact with endoplasmic reticulum membrane adaptor (ERAdP) and conferred anti-bacterial immunity in mice through the production of proinflammatory mediators.⁹ The very similar mechanism of immune system activation by the detection of cyclic dinucleotides (CDNs) in murine cells led us to hypothesize that signaling proteins other than STING might serve as receptors for cGAMP to trigger biological functions in human cells.

To address this hypothesis, we used cells of the human monocytic lineage THP-1 in which STING expression was abolished using CRISPR/Cas9 technology (STING KO). Both wild-type (WT) and STING KO THP-1 cells were differentiated into macrophages using PMA treatment and were subsequently challenged with cGAMP for 6 h. As previously validated,¹⁰ STING-deficient (STING-KO) THP-1 cells have lost the ability to induce type I IFN in response to stimulation with cytosolic DNA but have retained the response to stimulation with cytosolic RNA (Poly (I:C)) (Fig. 1a).

Fig. 1.

Global transcriptional responses to cGAMP stimulation depend on STING. a PMA differentiated WT (TMEM173^+/+) and STING KO (TMEM173^−/−) THP-1 cells were either left untreated (Nt) or stimualted with cGAMP (100 nM) or with poly I:C (4 μg mL⁻¹). Supernatants were collected after 20 h and analyzed for type I IFN using a HEK293 based assay. The data are representative of two independent experiments. Bars indicate mean and s.e.m. of three samples. b, c PMA differentiated WT (TMEM173^+/+) and STING KO (TMEM173^−/−) THP-1 cells were either left untreated (No treatment) or stimualted with cGAMP (100 nM) for 6 h. Cells were then lysed and analyzed by RNA sequencing. List of differentially regulated genes were indentified using 50-fold (b) and 5-fold (c) cut-offs at p value of 0.01 or less. Graphs display dendrograms where both differentially regualted genes and samples were clustered

To globally characterize the genes and signaling pathways affected by cGAMP stimulation in the absence of STING, we performed full transcriptome RNA sequencing analysis on cGAMP-stimulated WT and STING-KO THP-1 cells. As expected, the most strongly induced genes (>50-fold) in WT THP-1 cells comprised type I IFNs and a series of type I IFN stimulated genes (ISGs), including IFNB1, IFIT1, and CXCL10. As also expected, these genes were not induced in the absence of STING (Fig. 1b).

Surprisingly, the total dependency on STING was not limited to highly induced genes with a known connection to type I IFN immunology. When lowering the threshold to >5-fold, we identified only 119 genes that were differentially regulated by stimulation with cGAMP. Most of these genes were interferons, known ISGs or genes known to be inducible by the cGAS-STING pathway through downstream NFkB and/or IRF3 activation. Further, this regulation was completely dependent on STING, and we were therefore unable to identify genes or signaling pathways that were affected by cGAMP in the absence of STING in these cells (Fig. 1c).

Our failure to identify genes or pathways induced by cGAMP in a STING-independent manner strongly suggests that STING is the sole receptor for cGAMP to induce transcriptional changes in THP-1 cells. Our findings do not, however, exclude the existence of cGAMP receptor(s) that exert their function(s) through post-translational mechanisms and whose effects therefore did not appear in full-transcriptome analysis. Although we find it surprising that cGAMP was unable to induce global transcriptional changes in a STING-independent manner, this finding is in line with the absence of reports on other genes with significant structural or sequence homology to STING. The resulting non-redundancy of STING makes it a candidate target by pathogens seeking to avoid innate immune responses. Further, it highlights STING as a non-redundant target in the cGAS-STING pathway for pharmaceutical intervention in diseases in which excessive amounts of self-DNA drive a cGAS-STING-dependent inflammatory condition.

Competing interests

The authors declare no competing interests.