Molecular evolutionary and structural analysis of the cytosolic DNA sensor cGAS and STING

Cyclic GMP-AMP (cGAMP) synthase (cGAS) is recently identified as a cytosolic DNA sensor and generates a non-canonical cGAMP that contains G(2′,5′)pA and A(3′,5′)pG phosphodiester linkages. cGAMP activates STING which triggers innate immune responses in mammals. However, the evolutionary functions and origins of cGAS and STING remain largely elusive. Here, we carried out comprehensive evolutionary analyses of the cGAS-STING pathway. Phylogenetic analysis of cGAS and STING families showed that their origins could be traced back to a choanoflagellate Monosiga brevicollis. Modern cGAS and STING may have acquired structural features, including zinc-ribbon domain and critical amino acid residues for DNA binding in cGAS as well as carboxy terminal tail domain for transducing signals in STING, only recently in vertebrates. In invertebrates, cGAS homologs may not act as DNA sensors. Both proteins cooperate extensively, have similar evolutionary characteristics, and thus may have co-evolved during metazoan evolution. cGAS homologs and a prokaryotic dinucleotide cyclase for canonical cGAMP share conserved secondary structures and catalytic residues. Therefore, non-mammalian cGAS may function as a nucleotidyltransferase and could produce cGAMP and other cyclic dinucleotides. Taken together, assembling signaling components of the cGAS-STING pathway onto the eukaryotic evolutionary map illuminates the functions and origins of this innate immune pathway.     

Cancer cell-specific cGAS/STING Signaling pathway in the era of advancing cancer cell biology

Pattern-recognition receptors (PRRs) are critical to recognizing endogenous and exogenous threats to mount a protective proinflammatory innate immune response. PRRs may be located on the outer cell membrane, cytosol, and nucleus. The cGAS/STING signaling pathway is a cytosolic PRR system. Notably, cGAS is also present in the nucleus. The cGAS-mediated recognition of cytosolic dsDNA and its cleavage into cGAMP activates STING. Furthermore, STING activation through its downstream signaling triggers different interferon-stimulating genes (ISGs), initiating the release of type 1 interferons (IFNs) and NF-κB-mediated release of proinflammatory cytokines and molecules. Activating cGAS/STING generates type 1 IFN, which may prevent cellular transformation and cancer development, growth, and metastasis. The current article delineates the impact of the cancer cell-specific cGAS/STING signaling pathway alteration in tumors and its impact on tumor growth and metastasis. This article further discusses different approaches to specifically target cGAS/STING signaling in cancer cells to inhibit tumor growth and metastasis in conjunction with existing anticancer therapies.     

Autophagy side of MB21D1/cGAS DNA sensor

The MB21D1/cGAS (Mab-21 domain-containing 1/cyclic GMP-AMP [cGAMP] synthetase), acts as an intracellular pattern recognition receptor (PPR) to sense cytosolic pathogen DNAs and subsequently generates the second messenger cGAMP to initiate the TMEM173/STING pathway for interferon (IFN) production. Intriguingly, we have recently demonstrated crosstalk between the intracellular DNA sensing pathway and autophagy machinery by demonstrating a direct interaction between the MB21D1 DNA sensor and the BECN1/Beclin 1 autophagy protein. This interaction not only suppresses MB21D1 enzymatic activity to halt cGAMP production, but also enhances the autophagy-mediated degradation of cytosolic microbial DNAs. This demonstrates that MB21D1 is the molecular link between the intracellular DNA sensing pathway and the autophagy pathway, ultimately developing well-balanced immune responses against pathogens.     

Autophagy side of MB21D1/cGAS DNA sensor

The MB21D1/cGAS (Mab-21 domain-containing 1/cyclic GMP-AMP [cGAMP] synthetase), acts as an intracellular pattern recognition receptor (PPR) to sense cytosolic pathogen DNAs and subsequently generates the second messenger cGAMP to initiate the TMEM173/STING pathway for interferon (IFN) production. Intriguingly, we have recently demonstrated crosstalk between the intracellular DNA sensing pathway and autophagy machinery by demonstrating a direct interaction between the MB21D1 DNA sensor and the BECN1/Beclin 1 autophagy protein. This interaction not only suppresses MB21D1 enzymatic activity to halt cGAMP production, but also enhances the autophagy-mediated degradation of cytosolic microbial DNAs. This demonstrates that MB21D1 is the molecular link between the intracellular DNA sensing pathway and the autophagy pathway, ultimately developing well-balanced immune responses against pathogens.     

cGAS Senses Human Cytomegalovirus and Induces Type I Interferon Responses in Human Monocyte-Derived Cells

Human cytomegalovirus (HCMV) infections of healthy individuals are mostly unnoticed and result in viral latency. However, HCMV can also cause devastating disease, e.g., upon reactivation in immunocompromised patients. Yet, little is known about human immune cell sensing of DNA-encoded HCMV. Recent studies indicated that during viral infection the cyclic GMP/AMP synthase (cGAS) senses cytosolic DNA and catalyzes formation of the cyclic di-nucleotide cGAMP, which triggers stimulator of interferon genes (STING) and thus induces antiviral type I interferon (IFN-I) responses. We found that plasmacytoid dendritic cells (pDC) as well as monocyte-derived DC and macrophages constitutively expressed cGAS and STING. HCMV infection further induced cGAS, whereas STING expression was only moderately affected. Although pDC expressed particularly high levels of cGAS, and the cGAS/STING axis was functional down-stream of STING, as indicated by IFN-I induction upon synthetic cGAMP treatment, pDC were not susceptible to HCMV infection and mounted IFN-I responses in a TLR9-dependent manner. Conversely, HCMV infected monocyte-derived cells synthesized abundant cGAMP levels that preceded IFN-I production and that correlated with the extent of infection. CRISPR/Cas9- or siRNA-mediated cGAS ablation in monocytic THP-1 cells and primary monocyte-derived cells, respectively, impeded induction of IFN-I responses following HCMV infection. Thus, cGAS is a key sensor of HCMV for IFN-I induction in primary human monocyte-derived DC and macrophages.     

Building unique bonds to fight misplaced DNA

A cyclic dinucleotide comprised of GMP and AMP was previously shown to be a key intermediate during activation of innate immune responses to cytosolic DNA. A report by Patel and Tuschl groups published in Cell reveals the structure of the enzyme involved in the synthesis of this second messenger and identifies this cyclic dinucleotide as a unique compound in metazoan cell signaling.For more than 100 years it has been known that DNA stimulates immune responses¹. Hence, when DNA reaches the cytoplasmic compartment in a cell, no matter originating from an infectious agent like viruses or from the damaged nucleus or mitochondria, it is recognized as a sign of danger. DNA can provoke severe consequences as it can be seen from aberrant recognition of lost DNA in autoimmune conditions such as systemic lupus erythematous and Sjogren''s syndrome. To perceive such a dreadful insult, several DNA-sensing proteins are present in mammalian cells. Some of these DNA sensors activate a cytoplasmic protein called stimulator of interferon (IFN) genes (STING). STING then turns on a series of protein kinases, culminating in the production of type I IFNs and other cytokines that participate in host immune responses². Gaining details about the structures and the mechanisms associated with such cellular responses has been a matter of great interest in the immunology field and may bear relevance for both infectious and autoimmune conditions.It was recently demonstrated that STING activation by DNA is mediated by a cyclic dinucleotide comprised of GMP and AMP, called cGAMP. Hence, upon infection with DNA viruses or delivery of DNA into the cytoplasm of some immune cells, cGAMP levels build up, and the dinucleotide binds directly to STING, leading to type I IFN production through activation of IRF3 via TBK1³. Therefore, cGAMP acts as a second messenger during DNA-triggered innate immune response. It was also shown that cGAMP synthesis relies on the activity of the enzyme cyclic GMP-AMP synthase (cGAS), which belongs to the nucleotidyltransferase family⁴. cGAS, therefore, acts as a cytoplasmic DNA sensor that generates the second messenger cGAMP, essential for activating STING-mediated type I IFN production.Cyclic dinucleotides are well-known bacterial intracellular signal transducers, and cyclic di-GMP (c-di-GMP) has been acknowledged as a universal bacterial second messenger⁵. The structural and biochemical analysis of the bacterial enzymes responsible for the synthesis of this second messenger suggested that c-di-GMP is formed from two molecules of GTP via a two-step reaction that generates a 3′-5′-phosphodiester linkage between the two GMP nucleotides⁶. Taking the bacterial synthesis as a model and based on the fact that chemically synthesized cGAMP with the 3′-5′-phosphodiester linkage stimulates STING-dependent type I IFN production in mammalian cells³, one would assume that cGAS-derived cGAMP likely contains the same phosphodiester linkage. However, in an outstanding paper published by Cell, Gao et al.⁷ challenged this view. Combining structural, chemical, biochemical and biological techniques, they definitely establish that cGAMP contains a 2′-5′ linkage, position this second messenger as the first 2′-5′ linkage-containing metazoan second messenger ever described, and distinguish it from the bacterial cyclic dinucleotides. The previous study had concluded that the form of cGAMP generated in mammalian cells was a 3′-5′-phosphodiester nucleotide. In this study, however, Gao et al. identify cGAMP as actually cyclic [G(2′,5′)pA(3′,5′)p] cGAMP. This form is unique to metazoans. The bacterial form is therefore subtly different and is less potent as an activator of STING³.As a first approach for understanding the mechanisms involved in cGAMP synthesis after DNA recognition, the authors compared the structure of the crystalized cGAS in its free state with the structure of the enzyme complexed with double-stranded DNA (dsDNA). dsDNA interaction with the enzyme led to pronounced conformational changes on the protein, allowing cGAS to adopt a catalytically competent conformation, a feature considered to be essential for a cytosolic DNA sensor. Comparison of the structures of the dsDNA-bound cGAS complexed with GTP, or with GMP + ATP or with GTP + ATP suggested that one of the phosphodiester linkages in the dinucleotide produced by the reaction was of the 2′-5′ nature, in contrast to the previously assumed 3′-5′ conformation. This unexpected result was supported by biochemical analysis and confirmed after comparison of the purified cGAS-derived product with chemically synthesized dinucleotide standards.The authors have also provided evidence suggesting that cyclization occurs in a stepwise manner and showed that a pair of divalent cations is necessary for phosphodiester bond formation. Finally, the use of functional mutants of the dsDNA-binding site or of the catalytic pocket of cGAS reinforced the conclusions gained from the structural analysis, confirming the importance of complex formation between cGAS and dsDNA and of the nucleotide-interacting residues in the catalytic pocket for activity of cGAS and consequent STING-mediated type I IFN production.The great amount of data presented by Gao et al. provide detailed information regarding the synthesis of cGAMP by cGAS, and valuable knowledge for understanding the control of cellular responses to cytosolic DNA (Figure 1). Although 2′-5′ bonds were previously shown to occur in mammalian biochemical reactions during the polymerization of ATP into linear oligoadenilate by the dsRNA sensor oligoadenylate synthetase 1 (OAS1)⁸, this is the first documented case of such a linkage in dinucleotides. This kind of phosphodiester bond is uncommon, and few nucleases are reported to be able to hydrolyze 2′-5′ linkages⁹. This might promote a greater stability of the second messenger in cells and consequently enable effective and, maybe, long-lasting signal transduction. This unique structure establishes cGAMP as a founding member of a potentially broader class of metazoan second messengers. Importantly, the fact that the second messenger and the enzymes involved in dinucleotide synthesis in bacterial systems present some structural distinctions from the ones found in metazoan cells points to features that may be explored for selectively targeting the prokaryotic or the metazoan pathway. In addition, although it was not clearly demonstrated that cGAS and cGAMP directly impact in autoimmune responses, the structural and biochemical information provided by Gao et al. may bear relevance for the development of small molecule inhibitors with therapeutic potentials in such conditions.Open in a separate windowFigure 1The study by Gao et al. shows that, upon DNA recognition in cytoplasm, cGAS suffers a conformational shift that allows it to convert GTP and ATP nucleotides into the cyclic compound cGAMP, containing the [G(2′,5′)pA(3′,5′)p] linkages. The 2′-5′ linkage is a unique feature of metazoan cyclic dinucleotides, as bacterial ones described so far present exclusively 3′-5′ phosphodiester bonds. cGAMP subsequently binds to STING, leading to TBK1-mediated IRF3 activation and robust type I IFN production.     

Discovery of IACS-8803 and IACS-8779, potent agonists of stimulator of interferon genes (STING) with robust systemic antitumor efficacy

Activation of the stimulator of interferon genes (STING) pathway by both exogenous and endogenous cytosolic DNA results in the production of interferon beta (IFN-β) and is required for the generation of cytotoxic T-cell priming against tumor antigens. In the clinical setting, pharmacological stimulation of the STING pathway has the potential to synergize with immunotherapy antibodies by boosting anti-tumor immune responses. We report the discovery of two highly potent cyclic dinucleotide STING agonists, IACS-8803 and IACS-8779, which show robust activation of the STING pathway in vitro and a superior systemic anti-tumor response in the B16 murine model of melanoma when compared to one of the clinical benchmark compounds.     

Cytosolic RNA:DNA hybrids activate the cGAS–STING axis

Intracellular recognition of non‐self and also self‐nucleic acids can result in the initiation of potent pro‐inflammatory and antiviral cytokine responses. Most recently, cGAS was shown to be critical for the recognition of cytoplasmic dsDNA. Binding of dsDNA to cGAS results in the synthesis of cGAMP(2′–5′), which then binds to the endoplasmic reticulum resident protein STING. This initiates a signaling cascade that triggers the induction of an antiviral immune response. While most studies on intracellular nucleic acids have focused on dsRNA or dsDNA, it has remained unexplored whether cytosolic RNA:DNA hybrids are also sensed by the innate immune system. Studying synthetic RNA:DNA hybrids, we indeed observed a strong type I interferon response upon cytosolic delivery of this class of molecule. Studies in THP‐1 knockout cells revealed that the recognition of RNA:DNA hybrids is completely attributable to the cGAS–STING pathway. Moreover, in vitro studies showed that recombinant cGAS produced cGAMP upon RNA:DNA hybrid recognition. Altogether, our results introduce RNA:DNA hybrids as a novel class of intracellular PAMP molecules and describe an alternative cGAS ligand next to dsDNA.     

RNF111-facilitated neddylation potentiates cGAS-mediated antiviral innate immune response

The cytosolic DNA sensor cyclic GMP-AMP (cGAMP) synthetase (cGAS) has emerged as a fundamental component fueling the anti-pathogen immunity. Because of its pivotal role in initiating innate immune response, the activity of cGAS must be tightly fine-tuned to maintain immune homeostasis in antiviral response. Here, we reported that neddylation modification was indispensable for appropriate cGAS-STING signaling activation. Blocking neddylation pathway using neddylation inhibitor MLN4924 substantially impaired the induction of type I interferon and proinflammatory cytokines, which was selectively dependent on Nedd8 E2 enzyme Ube2m. We further found that deficiency of the Nedd8 E3 ligase Rnf111 greatly attenuated DNA-triggered cGAS activation while not affecting cGAMP induced activation of STING, demonstrating that Rnf111 was the Nedd8 E3 ligase of cGAS. By performing mass spectrometry, we identified Lys231 and Lys421 as essential neddylation sites in human cGAS. Mechanistically, Rnf111 interacted with and polyneddylated cGAS, which in turn promoted its dimerization and enhanced the DNA-binding ability, leading to proper cGAS-STING pathway activation. In the same line, the Ube2m or Rnf111 deficiency mice exhibited severe defects in innate immune response and were susceptible to HSV-1 infection. Collectively, our study uncovered a vital role of the Ube2m-Rnf111 neddylation axis in promoting the activity of the cGAS-STING pathway and highlighted the importance of neddylation modification in antiviral defense.     

MITA/STING: A central and multifaceted mediator in innate immune response

The recognition of nucleic acids is a general strategy used by the host to detect invading pathogens. Many studies have established that MITA/STING is a central component in the innate immune response to cytosolic DNA and RNA derived from pathogens. MITA can act both as a direct sensor of cyclic dinucleotides (CDNs) and as an adaptor for the recruitment of downstream signaling components. In both roles, MITA is part of signaling cascades that orchestrate innate immune defenses against various pathogens, including viruses, bacteria and parasites. Here, we highlight recent studies that have uncovered the molecular mechanisms of MITA-mediated signal transduction and regulation, and discuss some notable issues that remain elusive.     

STING agonist diABZI induces PANoptosis and DNA mediated acute respiratory distress syndrome (ARDS)

Stimulator of interferon genes (STING) contributes to immune responses against tumors and may control viral infection including SARS-CoV-2 infection. However, activation of the STING pathway by airway silica or smoke exposure leads to cell death, self-dsDNA release, and STING/type I IFN dependent acute lung inflammation/ARDS. The inflammatory response induced by a synthetic non-nucleotide-based diABZI STING agonist, in comparison to the natural cyclic dinucleotide cGAMP, is unknown. A low dose of diABZI (1 µg by endotracheal route for 3 consecutive days) triggered an acute neutrophilic inflammation, disruption of the respiratory barrier, DNA release with NET formation, PANoptosis cell death, and inflammatory cytokines with type I IFN dependent acute lung inflammation. Downstream upregulation of DNA sensors including cGAS, DDX41, IFI204, as well as NLRP3 and AIM2 inflammasomes, suggested a secondary inflammatory response to dsDNA as a danger signal. DNase I treatment, inhibition of NET formation together with an investigation in gene-deficient mice highlighted extracellular DNA and TLR9, but not cGAS, as central to diABZI-induced neutrophilic response. Therefore, activation of acute cell death with DNA release may lead to ARDS which may be modeled by diABZI. These results show that airway targeting by STING activator as a therapeutic strategy for infection may enhance lung inflammation with severe ARDS. Open in a separate windowSTING agonist diABZI induces neutrophilic lung inflammation and PANoptosis A, Airway STING priming induce a neutrophilic lung inflammation with epithelial barrier damage, double-stranded DNA release in the bronchoalvelolar space, cell death, NETosis and type I interferon release. B, 1. The diamidobenzimidazole (diABZI), a STING agonist is internalized into the cytoplasm through unknown receptor and induce the activation and dimerization of STING followed by TBK1/IRF3 phosporylation leading to type I IFN response. STING activation also leads to NF-kB activation and the production of pro-inflammatory cytokines TNFα and IL-6. 2. The activation of TNFR1 and IFNAR1 signaling pathway results in ZBP1 and RIPK3/ASC/CASP8 activation leading to MLKL phosphorylation and necroptosis induction. 3. This can also leads to Caspase-3 cleavage and apoptosis induction. 4. Self-dsDNA or mtDNA sensing by NLRP3 or AIM2 induces inflammsome formation leading to Gasdermin D cleavage enabling Gasdermin D pore formation and the release mature IL-1β and pyroptosis. NLRP3 inflammasome formation can be enhanced by the ZBP1/RIPK3/CASP8 complex. 5. A second signal of STING activation with diABZI induces cell death and the release of self-DNA which is sensed by cGAS and form 2′3′-cGAMP leading to STING hyper activation, the amplification of TBK1/IRF3 and NF-kB pathway and the subsequent production of IFN-I and inflammatory TNFα and IL-6. This also leads to IFI204 and DDX41 upregulation thus, amplifying the inflammatory loop. The upregulation of apoptosis, pyroptosis and necroptosis is indicative of STING-dependent PANoptosis. Subject terms: Cell death and immune response, Respiratory tract diseases     

Balancing STING in antimicrobial defense and autoinflammation

Rapid detection of microbes is crucial for eliciting an effective immune response. Innate immune receptors survey the intracellular and extracellular environment for signs of a microbial infection. When they detect a pathogen-associated molecular pattern (PAMP), such as viral DNA, they alarm the cell about the ongoing infection. The central signaling hub in sensing of viral DNA is the stimulator of interferon genes (STING). Upon activation, STING induces downstream signaling events that ultimately result in the production of type I interferons (IFN I), important cytokines in antimicrobial defense, in particular towards viruses. In this review, we describe the molecular features of STING, including its upstream sensors and ligands, its sequence and structural conservation, common polymorphisms, and its localization. We further highlight how STING activation requires a careful balance: its activity is essential for antiviral defense, but unwanted activation through mutations or accidental recognition of self-derived DNA causes autoinflammatory diseases. Several mechanisms, such as post-translational modifications, ensure this balance by fine-tuning STING activation. Finally, we discuss how viruses evade detection of their genomes by either exploiting cells that lack a functional DNA sensing pathway as a niche or by interfering with STING activation through viral evasion molecules. Insight into STING’s exact mechanisms in health and disease will guide the development of novel clinical interventions for microbial infections, autoinflammatory diseases, and beyond.     

Second messengers and divergent HD-GYP phosphodiesterases regulate 3′,3′-cGAMP signaling

3′,3′-cyclic GMP-AMP (cGAMP) is the third cyclic dinucleotide (CDN) to be discovered in bacteria. No activators of cGAMP signaling have yet been identified, and the signaling pathways for cGAMP have been inferred to display a narrow distribution based upon the characterized synthases, DncV and Hypr GGDEFs. Here, we report that the ubiquitous second messenger cyclic AMP (cAMP) is an activator of the Hypr GGDEF enzyme GacB from Myxococcus xanthus. Furthermore, we show that GacB is inhibited directly by cyclic di-GMP, which provides evidence for cross-regulation between different CDN pathways. Finally, we reveal that the HD-GYP enzyme PmxA is a cGAMP-specific phosphodiesterase (GAP) that promotes resistance to osmotic stress in M. xanthus. A signature amino acid change in PmxA was found to reprogram substrate specificity and was applied to predict the presence of non-canonical HD-GYP phosphodiesterases in many bacterial species, including phyla previously not known to utilize cGAMP signaling.     

DNA sensor cGAS-mediated immune recognition

The host takes use of pattern recognition receptors (PRRs) to defend against pathogen invasion or cellular damage. Among microorganism-associated molecular patterns detected by host PRRs, nucleic acids derived from bacteria or viruses are tightly supervised, providing a fundamental mechanism of host defense. Pathogenic DNAs are supposed to be detected by DNA sensors that induce the activation of NFκB or TBK1-IRF3 pathway. DNA sensor cGAS is widely expressed in innate immune cells and is a key sensor of invading DNAs in several cell types. cGAS binds to DNA, followed by a conformational change that allows the synthesis of cyclic guanosine monophosphate–adenosine monophosphate (cGAMP) from adenosine triphosphate and guanosine triphosphate. cGAMP is a strong activator of STING that can activate IRF3 and subsequent type I interferon production. Here we describe recent progresses in DNA sensors especially cGAS in the innate immune responses against pathogenic DNAs.     

Single Nucleotide Polymorphisms of Human STING Can Affect Innate Immune Response to Cyclic Dinucleotides

The STING (stimulator of interferon genes) protein can bind cyclic dinucleotides to activate the production of type I interferons and inflammatory cytokines. The cyclic dinucleotides can be bacterial second messengers c-di-GMP and c-di-AMP, 3’5’-3’5’ cyclic GMP-AMP (3’3’ cGAMP) produced by Vibrio cholerae and metazoan second messenger 2’5’-3’5’ Cyclic GMP-AMP (2’3’ cGAMP). Analysis of single nucleotide polymorphism (SNP) data from the 1000 Genome Project revealed that R71H-G230A-R293Q (HAQ) occurs in 20.4%, R232H in 13.7%, G230A-R293Q (AQ) in 5.2%, and R293Q in 1.5% of human population. In the absence of exogenous ligands, the R232H, R293Q and AQ SNPs had only modest effect on the stimulation of IFN-β and NF-κB promoter activities in HEK293T cells, while HAQ had significantly lower intrinsic activity. The decrease was primarily due to the R71H substitution. The SNPs also affected the response to the cyclic dinucleotides. In the presence of c-di-GMP, the R232H variant partially decreased the ability to activate IFN-βsignaling, while it was defective for the response to c-di-AMP and 3’3’ cGAMP. The R293Q dramatically decreased the stimulatory response to all bacterial ligands. Surprisingly, the AQ and HAQ variants maintained partial abilities to activate the IFN-β signaling in the presence of ligands due primarily to the G230A substitution. Biochemical analysis revealed that the recombinant G230A protein could affect the conformation of the C-terminal domain of STING and the binding to c-di-GMP. Comparison of G230A structure with that of WT revealed that the conformation of the lid region that clamps onto the c-di-GMP was significantly altered. These results suggest that hSTING variation can affect innate immune signaling and that the common HAQ haplotype expresses a STING protein with reduced intrinsic signaling activity but retained the ability to response to bacterial cyclic dinucleotides.     

Cyclic GMP-AMP Displays Mucosal Adjuvant Activity in Mice

The recently discovered mammalian enzyme cyclic GMP-AMP synthase produces cyclic GMP-AMP (cGAMP) after being activated by pathogen-derived cytosolic double stranded DNA. The product can stimulate STING-dependent interferon type I signaling. Here, we explore the efficacy of cGAMP as a mucosal adjuvant in mice. We show that cGAMP can enhance the adaptive immune response to the model antigen ovalbumin. It promotes antigen specific IgG and a balanced Th1/Th2 lymphocyte response in immunized mice. A characteristic of the cGAMP-induced immune response is the slightly reduced induction of interleukin-17 as a hallmark of Th17 activity – a distinct feature that is not observed with other cyclic di-nucleotide adjuvants. We further characterize the innate immune stimulation activity in vitro on murine bone marrow-derived dendritic cells and human dendritic cells. The observed results suggest the consideration of cGAMP as a candidate mucosal adjuvant for human vaccines.     

5,6-Dimethylxanthenone-4-acetic Acid (DMXAA) Activates Stimulator of Interferon Gene (STING)-dependent Innate Immune Pathways and Is Regulated by Mitochondrial Membrane Potential

The chemotherapeutic agent 5,6-dimethylxanthenone-4-acetic acid (DMXAA) is a potent inducer of type I IFNs and other cytokines. This ability is essential for its chemotherapeutic benefit in a mouse cancer model and suggests that it might also be useful as an antiviral agent. However, the mechanism underlying DMXAA-induced type I IFNs, including the host proteins involved, remains unclear. Recently, it was reported that the antioxidant N-acetylcysteine (NAC) decreased DMXAA-induced TNF-α and IL-6, suggesting that oxidative stress may play a role. The goal of this study was to identify host proteins involved in DMXAA-dependent signaling and determine how antioxidants modulate this response. We found that expression of IFN-β in response to DMXAA in mouse macrophages requires the mitochondrial and endoplasmic reticulum resident protein STING. Addition of the antioxidant diphenylene iodonium (DPI) diminished DMXAA-induced IFN-β, but this decrease was independent of both the NADPH oxidase, Nox2, and de novo generation of reactive oxygen species. Additionally, IFN-β up-regulation by DMXAA was inhibited by agents that target the mitochondrial electron transport chain and, conversely, loss of mitochondrial membrane potential correlated with diminished innate immune signaling in response to DMXAA. Up-regulation of Ifnb1 gene expression mediated by cyclic dinucleotides was also impaired by DPI, whereas up-regulation of Ifnb1 mRNA due to cytosolic double-stranded DNA was not. Although both stimuli signal through STING, cyclic dinucleotides interact directly with STING, suggesting that recognition of DMXAA by STING may also be mediated by direct interaction.