DNA sensing unchained

In two recent reports in Science, James Chen and colleagues provide compelling evidence that detection of cytosolic DNA triggers the production of a novel second messenger, cyclic GMP-AMP (cGAMP), which in turn activates a signaling pathway that induces type I interferons (IFNs) in a STING-dependent manner. They further unravel a key role for a so far uncharacterized murine protein E330016A19 (human homolog: C6ORF150), now termed cGAMP synthetase (cGAS), to act as the DNA sensor that generates cGAMP

An unexpected role for RNA in the recognition of DNA by the innate immune system

A central function of our innate immune system is to sense microbial pathogens through the presence of their nucleic acid genomes or their transcriptional or replicative activity. In mammals, a receptor-based system is mainly responsible for the detection of these "non self" nucleic acids. Tremendous progress has been made in the past years in identifying the host constituents that are required for this intricate task. With regard to the sensing of RNA genome based pathogens by our innate immune system, a picture is emerging that includes certain families of the toll-like receptor family (TLR3, TLR7, TLR8) and the RIG-I like helicases (RIG-I, MDA5 and LGP2). Genetic loss of function studies implicate that the absence of these pathways can lead to a complete lack of recognition of certain RNA viruses. At the same time, intracellular DNA can also trigger potent innate immune responses, yet the players in this field are less clear. We and another group have recently identified a role for RNA polymerase III in the conversion of AT-rich DNA into an RNA ligand that is sensed by the RIG-I pathway. In this review article, we will discuss the mechanisms and implications of this novel pathway

RIG-I-dependent sensing of poly(dA:dT) through the induction of an RNA polymerase III-transcribed RNA intermediate

RNA is sensed by Toll-like receptor 7 (TLR7) and TLR8 or by the RNA helicases LGP2, Mda5 and RIG-I to trigger antiviral responses. Much less is known about sensors for DNA. Here we identify a novel DNA-sensing pathway involving RNA polymerase III and RIG-I. In this pathway, AT-rich double-stranded DNA (dsDNA) served as a template for RNA polymerase III and was transcribed into double-stranded RNA (dsRNA) containing a 5'-triphosphate moiety. Activation of RIG-I by this dsRNA induced production of type I interferon and activation of the transcription factor NF-kappaB. This pathway was important in the sensing of Epstein-Barr virus-encoded small RNAs, which were transcribed by RNA polymerase III and then triggered RIG-I activation. Thus, RNA polymerase III and RIG-I are pivotal in sensing viral DNA

A mechanism for the inhibition of DNA-PK-mediated DNA sensing by a virus

The innate immune system is critical in the response to infection by pathogens and it is activated by pattern recognition receptors (PRRs) binding to pathogen associated molecular patterns (PAMPs). During viral infection, the direct recognition of the viral nucleic acids, such as the genomes of DNA viruses, is very important for activation of innate immunity. Recently, DNA-dependent protein kinase (DNA-PK), a heterotrimeric complex consisting of the Ku70/Ku80 heterodimer and the catalytic subunit DNA-PKcs was identified as a cytoplasmic PRR for DNA that is important for the innate immune response to intracellular DNA and DNA virus infection. Here we show that vaccinia virus (VACV) has evolved to inhibit this function of DNA-PK by expression of a highly conserved protein called C16, which was known to contribute to virulence but by an unknown mechanism. Data presented show that C16 binds directly to the Ku heterodimer and thereby inhibits the innate immune response to DNA in fibroblasts, characterised by the decreased production of cytokines and chemokines. Mechanistically, C16 acts by blocking DNA-PK binding to DNA, which correlates with reduced DNA-PK-dependent DNA sensing. The C-terminal region of C16 is sufficient for binding Ku and this activity is conserved in the variola virus (VARV) orthologue of C16. In contrast, deletion of 5 amino acids in this domain is enough to knockout this function from the attenuated vaccine strain modified vaccinia virus Ankara (MVA). In vivo a VACV mutant lacking C16 induced higher levels of cytokines and chemokines early after infection compared to control viruses, confirming the role of this virulence factor in attenuating the innate immune response. Overall this study describes the inhibition of DNA-PK-dependent DNA sensing by a poxvirus protein, adding to the evidence that DNA-PK is a critical component of innate immunity to DNA viruses

The cGAS-STING pathway drives type I IFN immunopathology in COVID-19.

COVID-19, which is caused by infection with SARS-CoV-2, is characterized by lung pathology and extrapulmonary complications <sup>1,2</sup> . Type I interferons (IFNs) have an essential role in the pathogenesis of COVID-19 (refs <sup>3-5</sup> ). Although rapid induction of type I IFNs limits virus propagation, a sustained increase in the levels of type I IFNs in the late phase of the infection is associated with aberrant inflammation and poor clinical outcome <sup>5-17</sup> . Here we show that the cyclic GMP-AMP synthase (cGAS)-stimulator of interferon genes (STING) pathway, which controls immunity to cytosolic DNA, is a critical driver of aberrant type I IFN responses in COVID-19 (ref. <sup>18</sup> ). Profiling COVID-19 skin manifestations, we uncover a STING-dependent type I IFN signature that is primarily mediated by macrophages adjacent to areas of endothelial cell damage. Moreover, cGAS-STING activity was detected in lung samples from patients with COVID-19 with prominent tissue destruction, and was associated with type I IFN responses. A lung-on-chip model revealed that, in addition to macrophages, infection with SARS-CoV-2 activates cGAS-STING signalling in endothelial cells through mitochondrial DNA release, which leads to cell death and type I IFN production. In mice, pharmacological inhibition of STING reduces severe lung inflammation induced by SARS-CoV-2 and improves disease outcome. Collectively, our study establishes a mechanistic basis of pathological type I IFN responses in COVID-19 and reveals a principle for the development of host-directed therapeutics

Listeria monocytogenes is sensed by the NLRP3 and AIM2 inflammasome

The inflammasome pathway functions to regulate caspase-1 activation in response to a broad range of stimuli. Caspase-1 activation is required for the maturation of the pivotal pro-inflammatory cytokines of the pro-IL-1beta family. In addition, caspase-1 activation leads to a certain type of cell death known as pyroptosis. Activation of the inflammasome has been shown to play a critical role in the recognition and containment of various microbial pathogens, including the intracellularly replicating Listeria monocytogenes; however, the inflammasome pathways activated during L. monocytogenes infection are only poorly defined. Here, we demonstrate that L. monocytogenes activates both the NLRP3 and the AIM2 inflammasome, with a predominant involvement of the AIM2 inflammasome. In addition, L. monocytogenes-triggered cell death was diminished in the absence of both AIM2 and NLRP3, and is concomitant with increased intracellular replication of L. monocytogenes. Altogether, these data establish a role for DNA sensing through the AIM2 inflammasome in the detection of intracellularly replicating bacteria

Transesophageal echocardiography-guided versus fluoroscopy-guided patent foramen ovale closure : A single center registry

Percutaneous closure of patent foramen ovale (PFO) is conventionally performed under continuous transesophageal echocardiographic (TEE) guidance. We aimed to evaluate whether a simplified procedural approach, including pure fluoroscopy-guidance and final TEE control, as well as an aimed 'next-day-discharge' is comparable with the conventional TEE-guided procedure in terms of periprocedural and intermediate-term outcomes.All patients who underwent a PFO closure at our center between 2010 and 2022 were retrospectively included. Prior to June 2019 cases were performed with continuous TEE guidance (TEE-guided group). Since June 2019, only pure fluoroscopy-guided PFO closures have been performed with TEE insertion and control just prior to device release (fluoroscopy-guided group). We analyzed procedural aspects, as well as long term clinical and echocardiographic outcomes.In total 291 patients were included in the analysis: 197 in the TEE-guided group and 94 in the fluoroscopy-guided group. Fluoroscopy-guided procedures were markedly shorter (48 ± 20 min vs. 25 ± 9 min; p < .01). There was no difference in procedural complications, including death, major bleeding, device dislodgement, stroke or clinically relevant peripheral embolization between the two groups (.5% vs. 0%; p = .99). Hospital stay was also shorter with the simplified approach (2.5 ± 1.6 vs. 3.5 ± 1.2 days; p < .01), allowing 85% same-day discharges during the last 12 months of observation period. At 6 ± 3 months echocardiographic follow-up a residual leakage was described in 8% of the TEE-guided cases and 2% of the fluoroscopy-guided cases (p = .08).While a complete TEE-free PFO closure might have potential procedural risks, our approach of pure fluoroscopy-guided with a brisk final TEE check seems to be advantageous in terms of procedural aspects with no sign of any acute or intermediate-term hazard and it could offer an equitable compromise between the two worlds: a complete TEE procedure and a procedure without any TEE

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-gamma, 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-beta 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