Andrea Ablasser

AIM2 recognizes cytosolic dsDNA and forms a caspase-1-activating inflammasome with ASC

The innate immune system senses nucleic acids by germline-encoded pattern recognition receptors. RNA is sensed by Toll-like receptor members TLR3, TLR7 and TLR8, or by the RNA helicases RIG-I (also known as DDX58) and MDA-5 (IFIH1). Little is known about sensors for cytoplasmic DNA that trigger antiviral and/or inflammatory responses. The best characterized of these responses involves activation of the TANK-binding kinase (TBK1)–interferon regulatory factor 3 (IRF3) signalling axis to trigger transcriptional induction of type I interferon genes. A second, less well-defined pathway leads to the activation of an ‘inflammasome’ that, via caspase-1, controls the catalytic cleavage of the pro-forms of the cytokines IL1β and IL18 (refs 6, 7). Using mouse and human cells, here we identify the PYHIN (pyrin and HIN domain-containing protein) family member absent in melanoma 2 (AIM2) as a receptor for cytosolic DNA, which regulates caspase-1. The HIN200 domain of AIM2 binds to DNA, whereas the pyrin domain (but not that of the other PYHIN family members) associates with the adaptor molecule ASC (apoptosis-associated speck-like protein containing a caspase activation and recruitment domain) to activate both NF-κB and caspase-1. Knockdown of Aim2 abrogates caspase-1 activation in response to cytoplasmic double-stranded DNA and the double-stranded DNA vaccinia virus. Collectively, these observations identify AIM2 as a new receptor for cytoplasmic DNA, which forms an inflammasome with the ligand and ASC to activate caspase-1.

Sequence-specific potent induction of IFN-alpha by short interfering RNA in plasmacytoid dendritic cells through TLR7

Short interfering RNA (siRNA) is used in RNA interference technology to avoid non-target-related induction of type I interferon (IFN) typical for long double-stranded RNA. Here we show that in plasmacytoid dendritic cells (PDC), an immune cell subset specialized in the detection of viral nucleic acids and production of type I IFN, some siRNA sequences, independent of their GU content, are potent stimuli of IFN-α production. Localization of the immunostimulatory motif on the sense strand of a potent IFN-α-inducing siRNA allowed dissection of immunostimulation and target silencing. Injection into mice of immunostimulatory siRNA, when complexed with cationic liposomes, induced systemic immune responses in the same range as the TLR9 ligand CpG, including IFN-α in serum and activation of T cells and dendritic cells in spleen. Immunostimulation by siRNA was absent in TLR7-deficient mice. Thus sequence-specific TLR7-dependent immune recognition in PDC needs to be considered as an additional biological activity of siRNA, which then should be termed immunostimulatory RNA (isRNA).

cGAS produces a 2′-5′-linked cyclic dinucleotide second messenger that activates STING

Detection of cytoplasmic DNA represents one of the most fundamental mechanisms of the innate immune system to sense the presence of microbial pathogens. Moreover, erroneous detection of endogenous DNA by the same sensing mechanisms has an important pathophysiological role in certain sterile inflammatory conditions. The endoplasmic-reticulum-resident protein STING is critically required for the initiation of type I interferon signalling upon detection of cytosolic DNA of both exogenous and endogenous origin. Next to its pivotal role in DNA sensing, STING also serves as a direct receptor for the detection of cyclic dinucleotides, which function as second messenger molecules in bacteria. DNA recognition, however, is triggered in an indirect fashion that depends on a recently characterized cytoplasmic nucleotidyl transferase, termed cGAMP synthase (cGAS), which upon interaction with DNA synthesizes a dinucleotide molecule that in turn binds to and activates STING. We here show in vivo and in vitro that the cGAS-catalysed reaction product is distinct from previously characterized cyclic dinucleotides. Using a combinatorial approach based on mass spectrometry, enzymatic digestion, NMR analysis and chemical synthesis we demonstrate that cGAS produces a cyclic GMP-AMP dinucleotide, which comprises a 2′-5′ and a 3′-5′ phosphodiester linkage >Gp(2′-5′)Ap(3′-5′)>. We found that the presence of this 2′-5′ linkage was required to exert potent activation of human STING. Moreover, we show that cGAS first catalyses the synthesis of a linear 2′-5′-linked dinucleotide, which is then subject to cGAS-dependent cyclization in a second step through a 3′-5′ phosphodiester linkage. This 13-membered ring structure defines a novel class of second messenger molecules, extending the family of 2′-5′-linked antiviral biomolecules.

Structural mechanism of cytosolic DNA sensing by cGAS

Cytosolic DNA arising from intracellular bacterial or viral infections is a powerful pathogen-associated molecular pattern (PAMP) that leads to innate immune host defence by the production of type I interferon and inflammatory cytokines. Recognition of cytosolic DNA by the recently discovered cyclic-GMP-AMP (cGAMP) synthase (cGAS) induces the production of cGAMP to activate the stimulator of interferon genes (STING). Here we report the crystal structure of cGAS alone and in complex with DNA, ATP and GTP along with functional studies. Our results explain the broad DNA sensing specificity of cGAS, show how cGAS catalyses dinucleotide formation and indicate activation by a DNA-induced structural switch. cGAS possesses a remarkable structural similarity to the antiviral cytosolic double-stranded RNA sensor 2′-5′oligoadenylate synthase (OAS1), but contains a unique zinc thumb that recognizes B-form double-stranded DNA. Our results mechanistically unify dsRNA and dsDNA innate immune sensing by OAS1 and cGAS nucleotidyl transferases.

Cell intrinsic immunity spreads to bystander cells via the intercellular transfer of cGAMP

The innate immune defence of multicellular organisms against microbial pathogens requires cellular collaboration. Information exchange allowing immune cells to collaborate is generally attributed to soluble protein factors secreted by pathogen-sensing cells. Cytokines, such as type I interferons (IFNs), serve to alert non-infected cells to the possibility of pathogen challenge. Moreover, in conjunction with chemokines they can instruct specialized immune cells to contain and eradicate microbial infection. Several receptors and signalling pathways exist that couple pathogen sensing to the induction of cytokines, whereas cytosolic recognition of nucleic acids seems to be exquisitely important for the activation of type I IFNs, master regulators of antiviral immunity. Cytosolic DNA is sensed by the receptor cyclic GMP-AMP (cGAMP) synthase (cGAS), which catalyses the synthesis of the second messenger cGAMP(2′-5′). This molecule in turn activates the endoplasmic reticulum (ER)-resident receptor STING, thereby inducing an antiviral state and the secretion of type I IFNs. Here we find in murine and human cells that cGAS-synthesized cGAMP(2′-5′) is transferred from producing cells to neighbouring cells through gap junctions, where it promotes STING activation and thus antiviral immunity independently of type I IFN signalling. In line with the limited cargo specificity of connexins, the proteins that assemble gap junction channels, most connexins tested were able to confer this bystander immunity, thus indicating a broad physiological relevance of this local immune collaboration. Collectively, these observations identify cGAS-triggered cGAMP(2′-5′) transfer as a novel host strategy that serves to rapidly convey antiviral immunity in a transcription-independent, horizontal manner.

Mycobacterium tuberculosis Differentially Activates cGAS- and Inflammasome-Dependent Intracellular Immune Responses through ESX-1

Cytosolic detection of microbial products is essential for the initiation of an innate immune response against intracellular pathogens such as Mycobacterium tuberculosis (Mtb). During Mtb infection of macrophages, activation of cytosolic surveillance pathways is dependent on the mycobacterial ESX-1 secretion system and leads to type I interferon (IFN) and interleukin-1β (IL-1β) production. Whereas the inflammasome regulates IL-1β secretion, the receptor(s) responsible for the activation of type I IFNs has remained elusive. We demonstrate that the cytosolic DNA sensor cyclic GMP-AMP synthase (cGAS) is essential for initiating an IFN response to Mtb infection. cGAS associates with Mtb DNA in the cytosol to stimulate cyclic GAMP (cGAMP) synthesis. Notably, activation of cGAS-dependent cytosolic host responses can be uncoupled from inflammasome activation by modulating the secretion of ESX-1 substrates. Our findings identify cGAS as an innate sensor of Mtb and provide insight into how ESX-1 controls the activation of specific intracellular recognition pathways.

T Cell-Independent, TLR-Induced IL-12p70 Production in Primary Human Monocytes

IL-12p70 is a key cytokine for the induction of Th1 immune responses. IL-12p70 production in myeloid cells is thought to be strictly controlled by T cell help. In this work we demonstrate that primary human monocytes can produce IL-12p70 in the absence of T cell help. We show that human monocytes express TLR4 and TLR8 but lack TLR3 and TLR7 even after preincubation with type I IFN. Simultaneous stimulation of TLR4 and TLR8 induced IL-12p70 in primary human monocytes. IL-12p70 production in peripheral blood myeloid dendritic cells required combined stimulation of TLR7/8 ligands together with TLR4 or with TLR3 ligands. In the presence of T cell-derived IL-4, but not IFN-γ, stimulation with TLR7/8 ligands was sufficient to stimulate IL-12p70 production. In monocytes, type I IFN was required but not sufficient to costimulate IL-12p70 induction by TLR8 ligation. Furthermore, TLR8 ligation inhibited LPS-induced IL-10 in monocytes, and LPS alone gained the ability to stimulate IL-12p70 in monocytes when the IL-10 receptor was blocked. Together, these results demonstrate that monocytes are licensed to synthesize IL-12p70 through type I IFN provided via the Toll/IL-1R domain-containing adaptor inducing IFN-β pathway and the inhibition of IL-10, both provided by combined stimulation with TLR4 and TLR8 ligands, triggering a potent Th1 response before T cell help is established.

OAS proteins and cGAS: unifying concepts in sensing and responding to cytosolic nucleic acids

Recent discoveries in the field of innate immunity have highlighted the existence of a family of nucleic acid-sensing proteins that have similar structural and functional properties. These include the well-known oligoadenylate synthase (OAS) family proteins and the recently identified OAS homologue cyclic GMP–AMP (cGAMP) synthase (cGAS). The OAS proteins and cGAS are template-independent nucleotidyltransferases that, once activated by double-stranded nucleic acids in the cytosol, produce unique classes of 2′–5′-linked second messenger molecules, which — through distinct mechanisms — have crucial antiviral functions. 2′–5′-linked oligoadenylates limit viral propagation through the activation of the enzyme RNase L, which degrades host and viral RNA, and 2′–5′-linked cGAMP activates downstream signalling pathways to induce de novo antiviral gene expression. In this Progress article, we describe the striking functional and structural similarities between OAS proteins and cGAS, and highlight their roles in antiviral immunity.

Selection of molecular structure and delivery of RNA oligonucleotides to activate TLR7 versus TLR8 and to induce high amounts of IL-12p70 in primary human monocytes

Detection of non-self RNA by TLRs within endosomes and by retinoic acid-inducible gene I (RIG-I)-like helicases in the cytosol is central to mammalian antiviral immunity. In this study, we used pathway-specific agonists and targeted delivery to address RNA immunorecognition in primary human immune cells. Within PBMC, plasmacytoid dendritic cells (pDC) and monocytes were found to be responsible for IFN-α production upon immunorecognition of RNA. The mechanisms of RNA recognition in pDC and monocytes were distinct. In pDC, recognition of ssRNA and dsRNA oligonucleotides was TLR7-dependent, whereas a 5′ triphosphate moiety (RIG-I ligand activity) had no major contribution to IFN-α production. In monocytes, the response to RNA oligonucleotides was mediated by either TLR8 or RIG-I. TLR8 was responsible for IL-12 induction upon endosomal delivery of ssRNA oligonucleotides and RIG-I was responsible for IFN-α production upon delivery of 5′ triphosphate RNA into the cytosol. In conclusion, the dissection of these pathways by selecting the appropriate structure and delivery of RNA reveals pDC as major producer of IFN-α upon TLR-mediated stimulation and monocytes as major producer of IFN-α upon RIG-I-mediated stimulation. Furthermore, our results uncover the potential of monocytes to function as major producers of IL-12p70, a key Th1 cytokine classically ascribed to myeloid dendritic cells that cannot be induced by CpG oligonucleotides in the human system.

Innate immune sensing of cytosolic chromatin fragments through cGAS promotes senescence

Cellular senescence is triggered by various distinct stresses and characterized by a permanent cell cycle arrest. Senescent cells secrete a variety of inflammatory factors, collectively referred to as the senescence-associated secretory phenotype (SASP). The mechanism(s) underlying the regulation of the SASP remains incompletely understood. Here we define a role for innate DNA sensing in the regulation of senescence and the SASP. We find that cyclic GMP-AMP synthase (cGAS) recognizes cytosolic chromatin fragments in senescent cells. The activation of cGAS, in turn, triggers the production of SASP factors via stimulator of interferon genes (STING), thereby promoting paracrine senescence. We demonstrate that diverse stimuli of cellular senescence engage the cGAS–STING pathway in vitro and we show cGAS-dependent regulation of senescence following irradiation and oncogene activation in vivo. Our findings provide insights into the mechanisms underlying cellular senescence by establishing the cGAS–STING pathway as a crucial regulator of senescence and the SASP.