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Type I interferon response, specifically, the cGAS-cGAMP-STING axis that results in IFN-β response, is well known for its complex roles early during viral infection. Previous reports suggest that HSV-1 DNA in Thp-1 cells and HIV-2 dsDNA in DCs and macrophages could be sensed by cGAS. The nuclear DNA sensor IFI16's viral DNA sensing leads to its acetylation, cytoplasmic translocation and STING activation and inflammasome activation. Although cGAS is known to be associated with IFI16 in the nucleus, however, during HSV-2 infection, the role of nuclear cGAS in viral DNA sensing, inflammasome formation and type I IFN response remains unknown. In the current study, extensive investigation of the complex IFN-β responses elicited early during HSV-2 infections in HFF cells is undertaken. The SiIFI16 and SicGAS treated HFF cells infected with HSV-2 demonstrate that cGAS senses nuclear herpes-viral DNA in an IFI16 dependent manner leading to nuclear cGAMP production. These results unravel a novel nuclear cooperative role of cGAS and IFI16 and extend the cGAS DNA sensing and its enzymatic activity in the nucleus. IFI16 acetylation required for inflammasome complex formation is cGAS independent. The cGAS-pro-Caspase1 and cGAS-ASC interaction suggests plausible role of cGAS in inflammasome complex for Caspase-1 activation. The activated Caspase-1 interaction with cGAS was also observed. Further, the autophagy and DNA damage responses elicited during HSV-2 infection are suggested. The crosstalk of the type I interferon pathway with the inflammasome, autophagy and DNA damage response pathways suggests an intricate mechanism of inter-regulation at different stages and time points during infection, that might orchestrate a balanced and efficient immune response or facilitate viral immune evasion. Unique and dynamic post translational modifications of cGAS, namely acetylation and K-63 poly-ubiquitination, are observed, and are plausibly involved in cGAS regulation during HSV-2 infection.

ObjectiveThe cGAS-STING pathway serves as a key mediator of inflammation. The aim of this study is to explore the biological role and molecular mechanisms of the cGAS-STING pathway in the fracture healing process, with a particular focus on its function during the early inflammatory phase.MethodsA murine femoral fracture model was utilized to investigate the activation of the cGAS-STING pathway during the early and late stages of bone healing. The methodologies encompassed transcriptome sequencing and immunohistochemistry. Pathway modulation was accomplished through the application of the STING inhibitor H-151 and the activator SR-717, with the effects being assessed via in vivo experiments (transcriptome sequencing, microCT, safranin O-fast green staining, and TRAP staining) and in vitro assays (TRAP staining, F-actin ring formation, bone resorption tests, qPCR, and Western blot analysis).ResultsTranscriptome analysis revealed a higher expression of STING on day 7 compared to day 21. Immunohistochemistry results also demonstrated significant activation of the cGAS-STING pathway within the fracture callus, particularly during the initial stages of healing. Transcriptome sequencing indicated that the activation of the cGAS-STING pathway by SR-717, in contrast to H-151, predominantly impacted osteoclasts while activating the NF-κB pathway. Results from microCT, safranin O-fast green, and TRAP staining suggested that the activation of the cGAS-STING pathway contributed to facilitating fracture healing and osteoclastogenesis. In vitro experiments further confirmed that SR-717 enhanced osteoclast formation and activity, while H-151 had an inhibitory effect. The underlying molecular mechanisms further demonstrated that the activation of the cGAS-STING pathway enhanced the transmission of the RANKL-induced NF-κB signaling pathway.ConclusionThe cGAS-STING pathway played a significant role in the early stages of healing in murine femoral fractures, accelerating the process of fracture repair. This pathway primarily influenced osteoclast differentiation and was associated with the key pathway of osteoclast formation, NF-κB.

Cellular senescence is historically regarded as a tumor suppression mechanism to prevent damaged cells from aberrant proliferation in benign and premalignant tumors. However, recent findings have suggested that senescent cells contribute to tumorigenesis and age‐associated pathologies through the senescence‐associated secretory phenotype (SASP). Therefore, to control age‐associated cancer, it is important to understand the molecular mechanisms of the SASP in the cancer microenvironment. New findings have suggested that the cyclic GMP‐AMP synthase (cGAS)‐stimulator of interferon genes (STING) signaling pathway, a critical indicator of innate immune response, triggers the SASP in response to accumulation of cytoplasmic DNA (cytoplasmic chromatin fragments, mtDNA and cDNA) in senescent cells. Notably, the cGAS‐STING signaling pathway promotes or inhibits tumorigenesis depending on the biological context in vivo, indicating that it may be a potential therapeutic target for cancer. Herein, we review the regulatory machinery and biological function of the SASP via the cGAS‐STING signaling pathway in cancer. This brief review discusses the role of the senescence‐associated secretory phenotype (SASP) and cyclic GMP‐AMP synthase‐stimulator of interferon genes (cGAS‐STING) signaling pathway on cellular senescence in cancer development. We suggest that the cGAS‐STING signaling pathway may be a novel target for induction of SASP.

The cyclic GMP-AMP synthase (cGAS)–cGAMP–STING pathway plays a key role in innate immunity, with cGAS sensing both pathogenic and mislocalized DNA in the cytoplasm. Human cGAS (h-cGAS) constitutes an important drug target for control of antiinflammatory responses that can contribute to the onset of autoimmune diseases. Recent studies have established that the positively charged N-terminal segment of cGAS contributes to enhancement of cGAS enzymatic activity as a result of DNA-induced liquid-phase condensation. We have identified an additional cGASCD–DNA interface (labeled site-C; CD, catalytic domain) in the crystal structure of a human SRY.cGASCD–DNA complex, with mutations along this basic site-C cGAS interface disrupting liquid-phase condensation, as monitored by cGAMP formation, gel shift, spin-down, and turbidity assays, as well as time-lapse imaging of liquid droplet formation. We expand on an earlier ladder model of cGAS dimers bound to a pair of parallel-aligned DNAs to propose a multivalent interaction-mediated cluster model to account for DNA-mediated condensation involving both the N-terminal domain of cGAS and the site-C cGAS–DNA interface. We also report the crystal structure of the h-cGASCD–DNA complex containing a triple mutant that disrupts the site-C interface, with this complex serving as a future platform for guiding cGAS inhibitor development at the DNA-bound h-cGAS level. Finally, we solved the structure of RU.521 bound in two alternate alignments to apo h-cGASCD, thereby occupying more of the catalytic pocket and providing insights into further optimization of active-site–binding inhibitors.

During Inflammaging, a dysregulation of the immune cell functions is generated, and these cells acquire a senescent phenotype with an increase in pro-inflammatory cytokines and ROS. This increase in pro-inflammatory molecules contributes to the chronic inflammation and oxidative damage of biomolecules, classically observed in the Inflammaging process. One of the most critical oxidative damages is generated to the host DNA. Damaged DNA is located out of the natural compartments, such as the nucleus and mitochondria, and is present in the cell’s cytoplasm. This DNA localization activates some DNA sensors, such as the cGAS/STING signaling pathway, that induce transcriptional factors involved in increasing inflammatory molecules. Some of the targets of this signaling pathway are the SASPs. SASPs are secreted pro-inflammatory molecules characteristic of the senescent cells and inducers of ROS production. It has been suggested that oxidative damage to nuclear and mitochondrial DNA generates activation of the cGAS/STING pathway, increasing ROS levels induced by SASPs. These additional ROS increase oxidative DNA damage, causing a loop during the Inflammaging. However, the relationship between the cGAS/STING pathway and the increase in ROS during Inflammaging has not been clarified. This review attempt to describe the potential connection between the cGAS/STING pathway and ROS during the Inflammaging process, based on the current literature, as a contribution to the knowledge of the molecular mechanisms that occur and contribute to the development of the considered adaptative Inflammaging process during aging.

Ataxia‐telangiectasia (A‐T) is a genetic disorder caused by the lack of functional ATM kinase. A‐T is characterized by chronic inflammation, neurodegeneration and premature ageing features that are associated with increased genome instability, nuclear shape alterations, micronuclei accumulation, neuronal defects and premature entry into cellular senescence. The causal relationship between the detrimental inflammatory signature and the neurological deficiencies of A‐T remains elusive. Here, we utilize human pluripotent stem cell‐derived cortical brain organoids to study A‐T neuropathology. Mechanistically, we show that the cGAS‐STING pathway is required for the recognition of micronuclei and induction of a senescence‐associated secretory phenotype (SASP) in A‐T olfactory neurosphere‐derived cells and brain organoids. We further demonstrate that cGAS and STING inhibition effectively suppresses self‐DNA‐triggered SASP expression in A‐T brain organoids, inhibits astrocyte senescence and neurodegeneration, and ameliorates A‐T brain organoid neuropathology. Our study thus reveals that increased cGAS and STING activity is an important contributor to chronic inflammation and premature senescence in the central nervous system of A‐T and constitutes a novel therapeutic target for treating neuropathology in A‐T patients. Aguado et al. show that senescent astrocytes upregulate detrimental pro‐inflammatory SASP factors in a cGAS/STING‐dependant manner that promote accelerated ageing in brain organoids of ataxia‐telangiectasia. Pharmacological interventions in these organoids with cGAS and STING inhibitors reduce astrocyte senescence and the SASP, and consequently, improve neuronal activity and survival.

Immune checkpoint blockade (ICB) has significantly advanced cancer immunotherapy, yet its patient response rates are generally low. Vaccines, including immunostimulant‐adjuvanted peptide antigens, can improve ICB. The emerging neoantigens generated by cancer somatic mutations elicit cancer‐specific immunity for personalized immunotherapy; the novel cyclic dinucleotide (CDN) adjuvants activate stimulator of interferon genes (STING) for antitumor type I interferon (IFN‐I) responses. However, CDN/neoantigen vaccine development has been limited by the poor antigen/adjuvant codelivery. Here, pH‐responsive CDN/neoantigen codelivering nanovaccines (NVs) for ICB combination tumor immunotherapy are reported. pH‐responsive polymers are synthesized to be self‐assembled into multivesicular nanoparticles (NPs) at physiological pH and disassembled at acidic conditions. NPs with high CDN/antigen coloading are selected as NVs for CDN/antigen codelivery to antigen presenting cells (APCs) in immunomodulatory lymph nodes (LNs). In the acidic endosome of APCs, pH‐responsive NVs facilitate the vaccine release and escape into cytosol, where CDNs activate STING for IFN‐I responses and antigens are presented by major histocompatibility complex (MHC) for T‐cell priming. In mice, NVs elicit potent antigen‐specific CD8+ T‐cell responses with immune memory, and reduce multifaceted tumor immunosuppression. In syngeneic murine tumors, NVs show robust ICB combination therapeutic efficacy. Overall, these CDN/neoantigen‐codelivering NVs hold the potential for ICB combination tumor immunotherapy. Cancer neoantigens and cyclic dinucleotide (CDN) vaccines can improve immune checkpoint blockade (ICB) immunotherapy, but have poor codelivery. Here, pH‐responsive nanovaccines are reported that efficiently codeliver CDNs/antigens to the cytosol of lymph nodal antigen presenting cells, resulting in efficient STING activation, durable antigen presentation, potent CD8+ T‐cell responses with memory, reduced tumor immunosuppression, and robust ICB combination tumor therapy.

The induction of type I interferons through the transcription factor interferon regulatory factor 3 (IRF3) is considered a major outcome of stimulator of interferon genes (STING) activation that drives immune responses against DNA viruses and tumors. However, STING activation can also trigger other downstream pathways such as nuclear factor κB (NF-κB) signaling and autophagy, and the roles of interferon (IFN)-independent functions of STING in infectious diseases or cancer are not well understood. Here, we generated a STING mouse strain with a mutation (S365A) that disrupts IRF3 binding and therefore type I interferon induction but not NF-κB activation or autophagy induction. We also generated STING mice with mutations that disrupt the recruitment of TANK-binding kinase 1 (TBK1), which is important for both IRF3 and NF-κB activation but not autophagy induction (L373A or ΔCTT, which lacks the C-terminal tail). The STING-S365A mutant mice, but not L373A or ΔCTT mice, were still resistant to herpes simplex virus 1 (HSV-1) infections and mounted an antitumor response after cyclic guanosine monophosphate-adenosine monophosphate (cGAMP) treatment despite the absence of STING-induced interferons. These results demonstrate that STING can function independently of type I interferons and autophagy, and that TBK1 recruitment to STING is essential for antiviral and antitumor immunity.

cGMP-AMP (cGAMP) synthase (cGAS) is a cytosolic DNA sensor that activates innate immune responses. cGAS catalyzes the synthesis of cGAMP, which functions as a second messenger that binds and activates the adaptor protein STING to induce type I interferons (IFNs) and other immune modulatory molecules. Here we show that cGAS is indispensable for the antitumor effect of immune checkpoint blockade in mice. Wild-type, but not cGAS-deficient, mice exhibited slower growth of B16 melanomas in response to a PD-L1 antibody treatment. Consistently, intramuscular delivery of cGAMP inhibited melanoma growth and prolonged the survival of the tumor-bearing mice. The combination of cGAMP and PD-L1 antibody exerted stronger antitumor effects than did either treatment alone. cGAMP treatment activated dendritic cells and enhanced cross-presentation of tumor-associated antigens to CD8 T cells. These results indicate that activation of the cGAS pathway is important for intrinsic antitumor immunity and that cGAMP may be used directly for cancer immunotherapy.

Burkholderia pseudomallei is the causative agent of melioidosis, an infectious disease in the tropics and subtropics with high morbidity and mortality. The facultative intracellular bacterium induces host cell fusion through its type VI secretion system 5 (T6SS5) as an important part of its pathogenesis in mammalian hosts. This allows it to spread intercellularly without encountering extracellular host defenses. We report that bacterial T6SS5-dependent cell fusion triggers type I IFN gene expression in the host and leads to activation of the cGAMP synthase–stimulator of IFN genes (cGAS–STING) pathway, independent of bacterial ligands. Aberrant and abortive mitotic events result in the formation of micronuclei colocalizing with cGAS, which is activated by double-stranded DNA. Surprisingly, cGAS–STING activation leads to type I IFN transcription but not its production. Instead, the activation of cGAS and STING results in autophagic cell death. We also observed type I IFN gene expression, micronuclei formation, and death of chemically induced cell fusions. Therefore, we propose that the cGAS–STING pathway senses unnatural cell fusion through micronuclei formation as a danger signal, and consequently limits aberrant cell division and potential cellular transformation through autophagic death induction.