cGAS is activated by DNA in a length‐dependent manner

Cytosolic DNA stimulates innate immune responses, including type I interferons (IFN), which have antiviral and immunomodulatory activities. Cyclic GMP‐AMP synthase (cGAS) recognizes cytoplasmic DNA and signals via STING to induce IFN production. Despite the importance of DNA in innate immunity, the nature of the DNA that stimulates IFN production is not well described. Using low DNA concentrations, we show that dsDNA induces IFN in a length‐dependent manner. This is observed over a wide length‐span of DNA, ranging from the minimal stimulatory length to several kilobases, and is fully dependent on cGAS irrespective of DNA length. Importantly, in vitro studies reveal that long DNA activates recombinant human cGAS more efficiently than short DNA, showing that length‐dependent DNA recognition is an intrinsic property of cGAS independent of accessory proteins. Collectively, this work identifies long DNA as the molecular entity stimulating the cGAS pathway upon cytosolic DNA challenge such as viral infections.     

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.     

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.     

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.     

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.     

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.     

Length does matter for cGAS

Recognition of foreign nucleic acids by the immune system is essential to host protection against many viral and bacterial infections. It relies on the capacity of innate immune sensors to selectively distinguish self‐ and non‐self‐nucleic acids, on the basis of a variety of parameters including base modifications, sequence composition, length or subcellular localisation. In this issue of EMBO Reports, Luecke et al 1 describe that the sensing of cytoplasmic double‐stranded DNA by the cyclic GMP–AMP (cGAMP) synthase (cGAS) is much more sensitive for longer fragments, when low doses of cytoplasmic DNA are used. This finding repositions length as the predominant factor governing the discrimination between self‐ and non‐self‐cytoplasmic DNA.     

RNA:DNA hybrids are a novel molecular pattern sensed by TLR9

The sensing of nucleic acids by receptors of the innate immune system is a key component of antimicrobial immunity. RNA:DNA hybrids, as essential intracellular replication intermediates generated during infection, could therefore represent a class of previously uncharacterised pathogen‐associated molecular patterns sensed by pattern recognition receptors. Here we establish that RNA:DNA hybrids containing viral‐derived sequences efficiently induce pro‐inflammatory cytokine and antiviral type I interferon production in dendritic cells. We demonstrate that MyD88‐dependent signalling is essential for this cytokine response and identify TLR9 as a specific sensor of RNA:DNA hybrids. Hybrids therefore represent a novel molecular pattern sensed by the innate immune system and so could play an important role in host response to viruses and the pathogenesis of autoimmune disease.     

Attenuation of cGAS‐STING signaling is mediated by a p62/SQSTM1‐dependent autophagy pathway activated by TBK1

Negative regulation of immune pathways is essential to achieve resolution of immune responses and to avoid excess inflammation. DNA stimulates type I IFN expression through the DNA sensor cGAS, the second messenger cGAMP, and the adaptor molecule STING. Here, we report that STING degradation following activation of the pathway occurs through autophagy and is mediated by p62/SQSTM1, which is phosphorylated by TBK1 to direct ubiquitinated STING to autophagosomes. Degradation of STING was impaired in p62‐deficient cells, which responded with elevated IFN production to foreign DNA and DNA pathogens. In the absence of p62, STING failed to traffic to autophagy‐associated vesicles. Thus, DNA sensing induces the cGAS‐STING pathway to activate TBK1, which phosphorylates IRF3 to induce IFN expression, but also phosphorylates p62 to stimulate STING degradation and attenuation of the response.     

Single Amino Acid Change in STING Leads to Constitutive Active Signaling

The production of cytokines by the immune system in response to cytosolic DNA plays an important role in host defense, autoimmune disease, and cancer immunogenicity. Recently a cytosolic DNA signaling pathway that is dependent on the endoplasmic reticulum adaptor and cyclic dinucleotide sensor protein STING has been identified. Association of cytosolic DNA with cyclic-GMP-AMP synthase (cGAS) activates its enzymatic activity to synthesize the cyclic dinucleotide second messenger cGAMP from GTP and ATP. Direct detection of cGAMP by STING triggers the activation of IRF3 and NF-kB, and the production of type I interferons and proinflammatory cytokines. The mechanism of how STING is able to mediate downstream signaling remains incompletely understood although it has been shown that dimerization is a prerequisite. Here, we identify a single amino acid change in STING that confers constitutive active signaling. This mutation appears to both enhance ability of STING to both dimerize and associate with its downstream target TBK1.     

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.     

RIG‐I detects infection with live Listeria by sensing secreted bacterial nucleic acids

Immunity against infection with Listeria monocytogenes is not achieved from innate immune stimulation by contact with killed but requires viable Listeria gaining access to the cytosol of infected cells. It has remained ill‐defined how such immune sensing of live Listeria occurs. Here, we report that efficient cytosolic immune sensing requires access of nucleic acids derived from live Listeria to the cytoplasm of infected cells. We found that Listeria released nucleic acids and that such secreted bacterial RNA/DNA was recognized by the cytosolic sensors RIG‐I, MDA5 and STING thereby triggering interferon β production. Secreted Listeria nucleic acids also caused RIG‐I‐dependent IL‐1β‐production and inflammasome activation. The signalling molecule CARD9 contributed to IL‐1β production in response to secreted nucleic acids. In conclusion, cytosolic recognition of secreted bacterial nucleic acids by RIG‐I provides a mechanistic explanation for efficient induction of immunity by live bacteria.     

Overlapping Patterns of Rapid Evolution in the Nucleic Acid Sensors cGAS and OAS1 Suggest a Common Mechanism of Pathogen Antagonism and Escape

A diverse subset of pattern recognition receptors (PRRs) detects pathogen-associated nucleic acids to initiate crucial innate immune responses in host organisms. Reflecting their importance for host defense, pathogens encode various countermeasures to evade or inhibit these immune effectors. PRRs directly engaged by pathogen inhibitors often evolve under recurrent bouts of positive selection that have been described as molecular ‘arms races.’ Cyclic GMP-AMP synthase (cGAS) was recently identified as a key PRR. Upon binding cytoplasmic double-stranded DNA (dsDNA) from various viruses, cGAS generates the small nucleotide secondary messenger cGAMP to signal activation of innate defenses. Here we report an evolutionary history of cGAS with recurrent positive selection in the primate lineage. Recent studies indicate a high degree of structural similarity between cGAS and 2’-5’-oligoadenylate synthase 1 (OAS1), a PRR that detects double-stranded RNA (dsRNA), despite low sequence identity between the respective genes. We present comprehensive comparative evolutionary analysis of cGAS and OAS1 primate sequences and observe positive selection at nucleic acid binding interfaces and distributed throughout both genes. Our data revealed homologous regions with strong signatures of positive selection, suggesting common mechanisms employed by unknown pathogen encoded inhibitors and similar modes of evasion from antagonism. Our analysis of cGAS diversification also identified alternately spliced forms missing multiple sites under positive selection. Further analysis of selection on the OAS family in primates, which comprises OAS1, OAS2, OAS3 and OASL, suggests a hypothesis where gene duplications and domain fusion events result in paralogs that provide another means of escaping pathogen inhibitors. Together our comparative evolutionary analysis of cGAS and OAS provides new insights into distinct mechanisms by which key molecular sentinels of the innate immune system have adapted to circumvent viral-encoded inhibitors.     

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.     

RIG-I Detects Triphosphorylated RNA of Listeria monocytogenes during Infection in Non-Immune Cells

The innate immune system senses pathogens by pattern recognition receptors in different cell compartments. In the endosome, bacteria are generally recognized by TLRs; facultative intracellular bacteria such as Listeria, however, can escape the endosome. Once in the cytosol, they become accessible to cytosolic pattern recognition receptors, which recognize components of the bacterial cell wall, metabolites or bacterial nucleic acids and initiate an immune response in the host cell. Current knowledge has been focused on the type I IFN response to Listeria DNA or Listeria-derived second messenger c-di-AMP via the signaling adaptor STING. Our study focused on the recognition of Listeria RNA in the cytosol. With the aid of a novel labeling technique, we have been able to visualize immediate cytosolic delivery of Listeria RNA upon infection. Infection with Listeria as well as transfection of bacterial RNA induced a type-I-IFN response in human monocytes, epithelial cells or hepatocytes. However, in contrast to monocytes, the type-I-IFN response of epithelial cells and hepatocytes was not triggered by bacterial DNA, indicating a STING-independent Listeria recognition pathway. RIG-I and MAVS knock-down resulted in abolishment of the IFN response in epithelial cells, but the IFN response in monocytic cells remained unaffected. By contrast, knockdown of STING in monocytic cells reduced cytosolic Listeria-mediated type-I-IFN induction. Our results show that detection of Listeria RNA by RIG-I represents a non-redundant cytosolic immunorecognition pathway in non-immune cells lacking a functional STING dependent signaling pathway.     

Innate immune sensing of nucleic acids from pathogens

The innate immune system is important as the first line of defense to sense invading pathogens. Nucleic acids represent critical pathogen signatures that trigger a host proinflammatory immune response. Much progress has been made in understanding how DNA and RNA trigger host defense countermeasures, however, several aspects of how cytosolic nucleic acids are sensed remain unclear. This special issue reviews how the host innate immune system senses nucleic acids from Brucella abortus, Mycobacterium sp and Legionella pneumophila, viral DNA, the role of STING in DNA sensing and inflammatory diseases and the mechanism of viral RNA recognition by the small interfering RNA pathway in Drosophila melanogaster.