cGAS Activation Research Articles

Disruption of mitochondrial dynamics triggers muscle inflammation through interorganellar contacts and mitochondrial DNA mislocation

Some forms of mitochondrial dysfunction induce sterile inflammation through mitochondrial DNA recognition by intracellular DNA sensors. However, the involvement of mitochondrial dynamics in mitigating such processes and their impact on muscle fitness remain unaddressed. Here we report that opposite mitochondrial morphologies induce distinct inflammatory signatures, caused by differential activation of DNA sensors TLR9 or cGAS. In the context of mitochondrial fragmentation, we demonstrate that mitochondria-endosome contacts mediated by the endosomal protein Rab5C are required in TLR9 activation in cells. Skeletal muscle mitochondrial fragmentation promotes TLR9-dependent inflammation, muscle atrophy, reduced physical performance and enhanced IL6 response to exercise, which improved upon chronic anti-inflammatory treatment. Taken together, our data demonstrate that mitochondrial dynamics is key in preventing sterile inflammatory responses, which precede the development of muscle atrophy and impaired physical performance. Thus, we propose the targeting of mitochondrial dynamics as an approach to treating disorders characterized by chronic inflammation and mitochondrial dysfunction.

INOS aggravates pressure overload-induced cardiac dysfunction via activation of the cytosolic-mtDNA-mediated cGAS-STING pathway.

Background: Sterile inflammation contributes to the pathogenesis of cardiac dysfunction caused by various conditions including pressure overload in hypertension. Mitochondrial DNA (mtDNA) released from damaged mitochondria has been implicated in cardiac inflammation. However, the upstream mechanisms governing mtDNA release and how mtDNA activates sterile inflammation in pressure-overloaded hearts remain largely unknown. Here, we investigated the role of inducible NO synthase (iNOS) on pressure overload-induced cytosolic accumulation of mtDNA and whether mtDNA activated inflammation through the cyclic GMP-AMP synthase (cGAS)-stimulator of interferon genes (STING) pathway. Methods: To investigate whether the cGAS-STING cascade was involved in sterile inflammation and cardiac dysfunction upon pressure overload, cardiomyocyte-specific STING depletion mice and mice injected with adeno-associated virus-9 (AAV-9) to suppress the cGAS-STING cascade in the heart were subjected to transverse aortic constriction (TAC). iNOS null mice were used to determine the role of iNOS in cGAS-STING pathway activation in pressure-stressed hearts. Results: iNOS knockout abrogated mtDNA release and alleviated cardiac sterile inflammation resulting in improved cardiac function. Conversely, activating the cGAS-STING pathway blunted the protective effects of iNOS knockout. Moreover, iNOS activated the cGAS-STING pathway in isolated myocytes and this was prevented by depleting cytosolic mtDNA. In addition, disruption of the cGAS-STING pathway suppressed inflammatory cytokine transcription and modulated M1/M2 macrophage polarization, and thus mitigated cardiac remodeling and improved heart function. Finally, increased iNOS expression along with cytosolic mtDNA accumulation and cGAS-STING activation were also seen in human hypertensive hearts. Conclusion: Our findings demonstrate that mtDNA is released into the cytosol and triggers sterile inflammation through the cGAS-STING pathway leading to cardiac dysfunction after pressure overload. iNOS controls mtDNA release and subsequent cGAS activation in pressure-stressed hearts.

Innate immune signaling drives late cardiac toxicity following DNA-damaging cancer therapies.

Late cardiac toxicity is a potentially lethal complication of cancer therapy, yet the pathogenic mechanism remains largely unknown, and few treatment options exist. Here we report DNA-damaging agents such as radiation and anthracycline chemotherapies inducing delayed cardiac inflammation following therapy due to activation of cGAS- and STING-dependent type I interferon signaling. Genetic ablation of cGAS-STING signaling in mice inhibits DNA damage-induced cardiac inflammation, rescues late cardiac functional decline, and prevents death from cardiac events. Treatment with a STING antagonist suppresses cardiac interferon signaling following DNA-damaging therapies and effectively mitigates cardiac toxicity. These results identify a therapeutically targetable, pathogenic mechanism for one of the most vexing treatment-related toxicities in cancer survivors.

Modulation of cGAS-STING signaling by PPARα in a mouse model of ischemia-induced retinopathy

In ischemic retinopathy, overactivated retinal myeloid cells are a crucial driving force of pathological angiogenesis and inflammation. The cyclic GMP-AMP synthase (cGAS) and stimulator of interferon genes (STING) signaling are key regulators of inflammation. This study aims to investigate the association of cGAS-STING signaling with ischemic retinopathy and the regulation of its activation. We found that protein levels of cGAS and STING were markedly up-regulated in retinal myeloid cells isolated from mice with oxygen-induced retinopathy (OIR). Knockout of Sting and pharmacological inhibition of STING both alleviated retinal neovascularization (NV) and reduced retinal vascular leakage in OIR. Further, Sting knockout and STING inhibitor also alleviated leukocyte adhesion to retinal vasculature and infiltration into the retina as well as microglial activation in OIR. These results suggest that cGAS-STING signaling played a pathogenic role in retinal myeloid cell activation and NV in ischemic retinopathy. To identify the regulation of cGAS-STING signaling in OIR, we evaluated the role of transcription factor peroxisome proliferator-activated receptor α (PPARα). The results demonstrated that PPARα was down-regulated in OIR retinas, primarily in myeloid cells. Furthermore, Pparα knockout significantly up-regulated cGAS and STING levels in retinal CD11b+ cells, while PPARα agonist inhibited cGAS-STING signaling and cytosolic mitochondrial DNA (mtDNA) release, a causative feature for cGAS activation. Knockout of Sting ameliorated retinal NV, hyperpermeability, and leukostasis in Pparα-/- mice with OIR. These observations suggest that PPARα regulates cGAS-STING signaling, likely through mtDNA release, and thus, is a potential therapeutic target for ischemic retinopathy.

DNA mechanical flexibility controls DNA potential to activate cGAS-mediated immune surveillance

DNA is well-documented to stimulate immune response. However, the nature of the DNA to activate immune surveillance is less understood. Here, we show that the activation of cyclic GMP-AMP synthase (cGAS) depends on DNA mechanical flexibility, which is controlled by DNA-sequence, -damage and -length. Consistently, DNA-sequence was shown to control cGAS activation. Structural analyses revealed that a conserved cGAS residue (mouse R222 or human R236) contributed to the DNA-flexibility detection. And the residue substitution neutralised the flexibility-controlled DNA-potential to activate cGAS, and relaxed the DNA-length specificity of cGAS. Moreover, low dose radiation was shown to mount cGAS-mediated acute immune surveillance (AIS) via repairable (reusable) DNAs in hrs. Loss of cGAS-mediated AIS decreased the regression of local and abscopal tumours in the context of focal radiation and immune checkpoint blockade. Our results build a direct link between immunosurveillance and DNA mechanical feature.

Regulation of cGAS activity by RNA-modulated phase separation.

Cyclic GMP-AMP synthase (cGAS) is a double-stranded DNA (dsDNA) sensor that functions in the innate immune system. Upon binding dsDNA, cGAS and dsDNA form phase-separated condensates in which cGAS catalyzes the synthesis of 2'3'-cyclic GMP-AMP that subsequently triggers a STING-dependent, type I interferon (IFN-I) response. Here, we show that cytoplasmic RNAs regulate cGAS activity. We discover that RNAs do not activate cGAS but rather promote phase separation of cGAS invitro. In cells, cGAS colocalizes with RNA and forms complexes with RNA. In the presence of cytoplasmic dsDNA, RNAs colocalize with phase-separated condensates of cGAS and dsDNA. Further invitro assays showed that RNAs promote the formation of cGAS-containing phase separations and enhance cGAS activity when the dsDNA concentration is low. Cotransfection of RNA with a small amount of dsDNA into THP1 cells significantly enhances the production of the downstream signaling molecule interferon beta (IFNB). This enhancement can be blocked by a cGAS-specific inhibitor. Thus, cytoplasmic RNAs could regulate cGAS activity by modulating the formation of cGAS-containing condensates.

Targeting SAMHD1 Promotes Anti-Tumor Immunity in Acute Myeloid Leukemia

The overall outcome of AML, which is characterized by HOXA9 overexpression (>50% cases), remains unfavorable. Success of allogeneic hematopoietic stem cell transplantation and immunotherapy highlights that T cell activation is critical for AML eradication. Notably, suppression of T cell immunity is common in the AML BM microenvironment, thus there is a pressing need to define underlying mechanisms. Recent studies reveal that the dNTP and Ara-CTP (active form of cytarabine) hydrolase SAMHD1, whose loss-of-function mutation is seen in inflammatory encephalopathy named Aicardi-Goutières syndrome, limits type-1 Interferon (IFN-I) and T cell responses by suppressing cGAS/STING signaling. Interestingly, our analysis of GEO datasets showed elevated SAMHD1 levels in specimens from AML patients relative to healthy donors, correlated with HOXA9 expression (A) and decreased T cell function. We thus asked whether SAMHD1 levels are associated with a particular cytogenetic change or mutation upstream of HOXA9 dysregulation. Accordingly, analysis of GSE data demonstrated that AML cases with MLL rearrangement (MLL-r, t[11q23]) or NPM1 mutation (NPM1c) exhibited elevated SAMHD1 levels relative to those without either alteration (B, C). Notably, HOXA9 knockdown (KD) decreased SAMHD1 protein levels in Molm13 (MLL-r+) and OCI-AML3 (NPM1c+) cells. ChIP-Q-PCR analysis of cord blood CD34+ cells engineered with Flag-tagged HoxA9 showed significant enrichment of HoxA9 protein at the predicted SAMHD1 promoter region relative to a control site. That region also showed significant enrichment of H3K27Ac (an active transcription mark) in HoxA9-overexpressing relative to control cells. We next assessed SAMHD1 loss-of-function in MLL-AF9 (MA9) knock-in mice, first by transducing leukemia cells from MA9+ BM with a doxycycline (DOX)-inducible control or shSAMHD1 and then transplanting them into either WT immunocompetent or immunodeficient (Rag2-/-) recipients to induce leukemia. Following DOX treatment, Rag2-/- mice bearing SAMHD1-KD or control transplants exhibited comparable survival. However, WT recipients bearing SAMHD1-KD AML survived significantly longer than controls (D, E). Moreover, SAMHD1-KD-mediated leukemia ablation was reproduced in HOXA9-Meis1 transduction/transplantation AML. Furthermore, depletion of T cells, but not B cells, blocked survival advantages seen in SAMHD1-KD-transplanted animals. To determine if IFN-I responses underlie these effects, we implanted SAMHD1-KD MA9 cells into WT or Ifnar1-/- recipients and observed significant abolition of SAMHD1-KD anti-AML effects. Moreover, SAMHD1-KD induced cGAS activation, as evidenced by elevated levels of the immune transmitter cGAMP in BM fluid from SAMHD1-KD relative to control MA9 mice. Accordingly, we observed upregulation of multiple interferon-stimulated genes (ISGs) such as ISG15 and CXCL10 downstream of cGAS/STING after SAMHD1-KD, effects blocked by cGAS deletion. To determine if immune activation requires SAMHD1 catalysis, we knocked out endogenous SAMHD1 in THP1 cells, which we then engineered cells to express doxycycline-inducible SAMHD1 variants including WT (SAMWT), catalytically-dead mutant SAMHD1 (SAMMT) or empty vector (MOCK). Comparison of ISGs expression in each group indicated that, relative to MOCK, SAMWT but not SAMMT downregulated ISGs, suggesting that blocking SAMHD1 catalysis promotes innate immunity. To identify SAMHD1 inhibitors, we conducted a structure-based virtual screen of 8,000,000 compounds (Molport). We then assessed the top 500 candidates using cell-based phenotypic screening based on cytarabine sensitization, namely, the inhibitor should sensitize SAMWT but not SAMMT cells to Ara-C toxicity (F). We then selected the 20 best candidates to evaluate for inhibition of SAMHD1 catalysis and chose Samin27 as the most potent lead compound. Importantly, we confirmed direct Samin27 interaction with SAMHD1 protein by nuclear magnetic resonance (NMR) (G). Samin27 treatment of SAMHD1-proficient human and mouse AML lines significantly upregulated ISGs expression. These results suggest that SAMHD1 plays an oncogenic role in AML by suppressing innate immunity. Further analysis is required to determine whether our compound can ablate AML in vivo when combined with currently available immune checkpoint inhibitors. Figure 1View largeDownload PPTFigure 1View largeDownload PPT Close modal

Targeting PRMT9 Elicits Cgas-Mediated Anti-Leukemia Immunity

Immunotherapy has improved outcomes for patients with blood cancers such as B-ALL. However, for patients with immune-cold cancers such as AML, currently available immunotherapies are less effective, likely due to lack of functional T cells and/or low expression of immune checkpoint proteins like PD-L1 . For these patients, development of an effective leukemia-targeting drug with immune-stimulating activity is required. We previously reported elevated PRMT9 levels in AML patient specimens, which enhanced leukemia cell proliferation by promoting RNA translation (Blood, 2021,138 [Supplement 1]: 358). Recently, our transcriptome profiling of AML lines revealed increased expression of interferon-stimulated genes (ISGs) after PRMT9-knockdown (KD), suggesting activation of innate immunity. To evaluate a potential PRMT9 function in anti-AML immunity, we transplanted either WT immunocompetent or Rag2-/- immunodeficient recipient mice with inducible Prmt9-KD or control MLL-AF9 (MA9) AML donor cells and monitored leukemia development by in-vivo bioluminescence Imaging. Unlike Rag2-/- recipients (overall survival <60 days), 5 of 7 WT recipients harboring Prmt9-KD AML survived the observation period (>120 days) and MA9 cells disappeared over time. To assess immune responses upon Prmt9 inhibition, we performed single-cell RNA-sequencing (scRNAseq) in BM and spleen cells from Prmt9-KD or control MA9-transplanted WT recipients (Fig. A). ScRNAseq analysis revealed that, relative to controls, Prmt9-KD induced T cell activation, based on increased T cell expression of Cd69, Gzmb and Ifng. After reanalyzing T cells at higher granularity based on levels of classical markers (e.g., Cd44, Sell, Fig. B, C), we observed reduced numbers of naïve T cells as well as increased effector/memory T cell subsets; increased numbers of cytotoxic T lymphocytes (CTLs) as well as decreased regulatory T cells (Tregs) subset (Fig. D). To validate T cell activation upon Prmt9 KD, we assessed an MA9/OVA transplant model (engineered with OVA to track antigen-specific T cells) and found that mice harboring Prmt9-KD MA9/OVA cells showed increased numbers of OVA-specific CD8+ T cells in BM tracked by flow cytometry after H-2Kb-OVA257-264 tetramer staining (Fig. E). Notably, scRNAseq analysis of multiple immune lineages in BM engrafted with Prmt9-KD MA9 cells revealed upregulation of Isg15, Cxcl10 and other ISGs. To confirm that type-I IFN responses mediate these effects, we implanted Prmt9-KD MA9 cells into WT or type-I IFN receptor KO (Ifnar1-/-) recipients and found that the survival advantage conferred by Prmt9-KD was lost in Ifnar1-/- recipients (Fig. F). Interestingly, Prmt9-KD in MA9 cells also increased expression of Isg15, Cxcl10 and other ISGs by Q-PCR assay, and the effect was blocked by cGAS deletion in MA9 cells, suggesting that Prmt9-KD activates cGAS-STING signaling. Finally, to determine whether Prmt9-KD phenotypes require tumor-intrinsic cGAS activity, we knocked out cGAS in Prmt9-KD MA9/OVA cells and implanted them into WT recipients. Relative to cGAS-WT controls, cGAS-KO in the presence of Prmt9-KD abolished survival advantages and cancer-specific T cell responses seen following Prmt9 KD in MA9/OVA cells (Fig. E). We also developed a potent new PRMT9 inhibitor (PRMT9i) based on our identified lead compound (Blood, 2021,138: 358). CyTOF analysis revealed that PRMT9i treatment ablated primary AML cells (CD34+CD45dim) and relatively increased T cell frequencies in co-cultures of immune cells with leukemia cells from primary AML specimens (n=3). However, PRMT9i anti-AML effects were significantly impaired by T cell depletion. Notably, CyTOF analysis revealed significantly upregulated PD-L1 levels in 2 of 3 (CD34+CD45dim) AML co-cultures after PRMT9i treatment (Fig. G), suggesting activation of the adaptive PD-1/PD-L1 axis, in agreement with the” unwanted negative effects” of cGAS-STING activation as others depicted. Accordingly, PRMT9i treatment of these 2 co-cultures combined with immune checkpoint inhibition (ICI, anti-PD1) synergistically eradicated primary AML cells (Fig. H) and promoted expansion of CD69+ and IFNγ+ CD8+ T cell subsets. Overall, we demonstrate that AML cell-derived PRMT9 regulates AML immune evasion, and that combining PRMT9i with ICIs could constitute a therapeutic strategy against PRMT9-proficient cancers like AML, in which single ICIs show limited efficacy. Figure 1View largeDownload PPTFigure 1View largeDownload PPT Close modal

Abstract 15420: Cgas-Sting Pathway-Mediated Cholesterol Metabolism Promotes Atherosclerosis Formation

Introduction: The mechanisms underlying macrophage lipid accumulation and foam cell formation, the critical step in atherosclerosis initiation, remain to be understood. The cGAS (cyclic GMP-AMP synthase)-STING (stimulator of interferon genes) signaling pathway triggers potent inflammation in response to cytosolic DNA accumulation. Hypothesis: In this study, we found that the cGAS-STING signaling pathway can promote macrophage lipid accumulation and atherosclerosis development. Methods: We developed an atherosclerosis mice model by a single intraperitoneal injection of an adeno-associated virus (AAV) containing a mouse PCSK9 gain-of-function mutation D377Y. Results: In the descending aorta of patients and aortic root of atherosclerotic mice, we detected profound cytosolic DNA accumulation that was associated with the activation of cGAS -STING in both human and mouse atherosclerotic plaques. In cultured macrophages, DNA uptake by macrophages activated the cGAS-STING pathway, which induced the biosynthesis of fatty acids and cholesterol. Interestingly, we found that STING can bind to SREBP2 (sterol regulatory element-binding protein 2) through SCAP (SREBP2 cleavage activating protein) and promote its cleavage and nuclear translocation, and subsequent induction of genes in fatty acid and cholesterol biosynthesis. Finally, in an AAV-PCSK9/high-fat diet-induced atherosclerosis model, cGas -/- and SMC- Sting -/- mice exhibited a reduction of SREBP2 activation and lipid content in macrophages and a reduction in atherosclerosis burden. Conclusions: Our findings indicated that DNA and cGAMP from damaged cells serve as a danger molecule that, by activating the cGAS-STING cytosolic DNA sensing signaling, promotes macrophage lipid biosynthesis and accumulation, contributing to atherosclerosis development.

Deficiency for SAMHD1 activates MDA5 in a cGAS/STING-dependent manner.

Defects in nucleic acid metabolizing enzymes can lead to spontaneous but selective activation of either cGAS/STING or RIG-like receptor (RLR) signaling, causing type I interferon-driven inflammatory diseases. In these pathophysiological conditions, activation of the DNA sensor cGAS and IFN production are linked to spontaneous DNA damage. Physiological, or tonic, IFN signaling on the other hand is essential to functionally prime nucleic acid sensing pathways. Here, we show that low-level chronic DNA damage in mice lacking the Aicardi-Goutières syndrome gene SAMHD1 reduced tumor-free survival when crossed to a p53-deficient, but not to a DNA mismatch repair-deficient background. Increased DNA damage did not result in higher levels of type I interferon. Instead, we found that the chronic interferon response in SAMHD1-deficient mice was driven by the MDA5/MAVS pathway but required functional priming through the cGAS/STING pathway. Our work positions cGAS/STING upstream of tonic IFN signaling in Samhd1-deficient mice and highlights an important role of the pathway in physiological and pathophysiological innate immune priming.

COVID-19 Molecular Pathophysiology: Acetylation of Repurposing Drugs

Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) induces immune-mediated type 1 interferon (IFN-1) production, the pathophysiology of which involves sterile alpha motif and histidine-aspartate domain-containing protein 1 (SAMHD1) tetramerization and the cytosolic DNA sensor cyclic-GMP-AMP synthase (cGAS)-stimulator of interferon genes (STING) signaling pathway. As a result, type I interferonopathies are exacerbated. Aspirin inhibits cGAS-mediated signaling through cGAS acetylation. Acetylation contributes to cGAS activity control and activates IFN-1 production and nuclear factor-κB (NF-κB) signaling via STING. Aspirin and dapsone inhibit the activation of both IFN-1 and NF-κB by targeting cGAS. We define these as anticatalytic mechanisms. It is necessary to alleviate the pathologic course and take the lag time of the odds of achieving viral clearance by day 7 to coordinate innate or adaptive immune cell reactions.

Evasion of cGAS and TRIM5 defines pandemic HIV

Of the 13 known independent zoonoses of simian immunodeficiency viruses to humans, only one, leading to human immunodeficiency virus (HIV) type 1(M) has become pandemic, causing over 80 million human infections. To understand the specific features associated with pandemic human-to-human HIV spread, we compared replication of HIV-1(M) with non-pandemic HIV-(O) and HIV-2 strains in myeloid cell models. We found that non-pandemic HIV lineages replicate less well than HIV-1(M) owing to activation of cGAS and TRIM5-mediated antiviral responses. We applied phylogenetic and X-ray crystallography structural analyses to identify differences between pandemic and non-pandemic HIV capsids. We found that genetic reversal of two specific amino acid adaptations in HIV-1(M) enables activation of TRIM5, cGAS and innate immune responses. We propose a model in which the parental lineage of pandemic HIV-1(M) evolved a capsid that prevents cGAS and TRIM5 triggering, thereby allowing silent replication in myeloid cells. We hypothesize that this capsid adaptation promotes human-to-human spread through avoidance of innate immune response activation.

FLASH X-ray spares intestinal crypts from pyroptosis initiated by cGAS-STING activation upon radioimmunotherapy

DNA-damaging treatments such as radiotherapy (RT) have become promising to improve the efficacy of immune checkpoint inhibitors by enhancing tumor immunogenicity. However, accompanying treatment-related detrimental events in normal tissues have posed a major obstacle to radioimmunotherapy and present new challenges to the dose delivery mode of clinical RT. In the present study, ultrahigh dose rate FLASH X-ray irradiation was applied to counteract the intestinal toxicity in the radioimmunotherapy. In the context of programmed cell death ligand-1 (PD-L1) blockade, FLASH X-ray minimized mouse enteritis by alleviating CD8+ T cell-mediated deleterious immune response compared with conventional dose rate (CONV) irradiation. Mechanistically, FLASH irradiation was less efficient than CONV X-ray in eliciting cytoplasmic double-stranded DNA (dsDNA) and in activating cyclic GMP-AMP synthase (cGAS) in the intestinal crypts, resulting in the suppression of the cascade feedback consisting of CD8+ T cell chemotaxis and gasdermin E-mediated intestinal pyroptosis in the case of PD-L1 blocking. Meanwhile, FLASH X-ray was as competent as CONV RT in boosting the antitumor immune response initiated by cGAS activation and achieved equal tumor control in metastasis burdens when combined with anti-PD-L1 administration. Together, the present study revealed an encouraging protective effect of FLASH X-ray upon the normal tissue without compromising the systemic antitumor response when combined with immunological checkpoint inhibitors, providing the rationale for testing this combination as a clinical application in radioimmunotherapy.

Caspase-Mediated Regulation and Cellular Heterogeneity of the cGAS/STING Pathway in Kaposi's Sarcoma-Associated Herpesvirus Infection.

As a result of the ongoing virus-host arms race, viruses have evolved numerous immune subversion strategies, many of which are aimed at suppressing the production of type I interferons (IFNs). Apoptotic caspases have recently emerged as important regulators of type I IFN signaling both in noninfectious contexts and during viral infection. Despite being widely considered antiviral factors since they can trigger cell death, several apoptotic caspases promote viral replication by suppressing innate immune response. Indeed, we previously discovered that the AIDS-associated oncogenic gammaherpesvirus Kaposi's sarcoma-associated herpesvirus (KSHV) exploits caspase activity to suppress the antiviral type I IFN response and promote viral replication. However, the mechanism of this novel viral immune evasion strategy is poorly understood, particularly with regard to how caspases antagonize IFN signaling during KSHV infection. Here, we show that caspase activity inhibits the DNA sensor cGAS during KSHV lytic replication to block type I IFN induction. Furthermore, we used single-cell RNA sequencing to reveal that the potent antiviral state conferred by caspase inhibition is mediated by an exceptionally small percentage of IFN-β-producing cells, thus uncovering further complexity of IFN regulation during viral infection. Collectively, these results provide insight into multiple levels of cellular type I IFN regulation that viruses co-opt for immune evasion. Unraveling these mechanisms can inform targeted therapeutic strategies for viral infections and reveal cellular mechanisms of regulating interferon signaling in the context of cancer and chronic inflammatory diseases. IMPORTANCE Type I interferons are key factors that dictate the outcome of infectious and inflammatory diseases. Thus, intricate cellular regulatory mechanisms are in place to control IFN responses. While viruses encode their own immune-regulatory proteins, they can also usurp existing cellular interferon regulatory functions. We found that caspase activity during lytic infection with the AIDS-associated oncogenic gammaherpesvirus Kaposi's sarcoma-associated herpesvirus inhibits the DNA sensor cGAS to block the antiviral type I IFN response. Moreover, single-cell RNA sequencing analyses unexpectedly revealed that an exceptionally small subset of infected cells (<5%) produce IFN, yet this is sufficient to confer a potent antiviral state. These findings reveal new aspects of type I IFN regulation and highlight caspases as a druggable target to modulate cGAS activity.

Endocytosis triggers V-ATPase-SYK–mediated priming of cGAS activation and innate immune response

The current view of nucleic acid-mediated innate immunity is that binding of intracellular sensors to nucleic acids is sufficient for their activation. Here, we report that endocytosis of virus or foreign DNA initiates a priming signal for the DNA sensor cyclic GMP-AMP synthase (cGAS)-mediated innate immune response. Mechanistically, viral infection or foreign DNA transfection triggers recruitment of the spleen tyrosine kinase (SYK) and cGAS to the endosomal vacuolar H+ pump (V-ATPase), where SYK is activated and then phosphorylates human cGASY214/215 (mouse cGasY200/201) to prime its activation. Upon binding to DNA, the primed cGAS initiates robust cGAMP production and mediator of IRF3 activation/stimulator of interferon genes-dependent innate immune response. Consistently, blocking the V-ATPase-SYK axis impairs DNA virus- and transfected DNA-induced cGAMP production and expression of antiviral genes. Our findings reveal that V-ATPase-SYK-mediated tyrosine phosphorylation of cGAS following endocytosis of virus or other cargos serves as a priming signal for cGAS activation and innate immune response.

USP15 promotes cGAS activation through deubiquitylation and liquid condensation.

Double-stranded DNA (dsDNA) is recognized as a danger signal by cyclic GMP-AMP synthase (cGAS), which triggers innate immune responses. cGAS activity must be properly regulated to maintain immune homeostasis. However, the mechanism by which cGAS activation is controlled remains to be better understood. In this study, we identified USP15 as a cGAS-interacting partner. USP15 promoted DNA-induced cGAS activation and downstream innate immune responses through a positive feedback mechanism. Specifically, USP15 deubiquitylated cGAS and promoted its activation. In the absence of DNA, USP15 drove cGAS dimerization and liquid condensation through the USP15 intrinsic disordered region (IDR), which prepared cGAS for a rapid response to DNA. Upon DNA stimulation, USP15 was induced to express and boost cGAS activation, functioning as an efficient amplifier in innate immune signal transduction. In summary, the positive role played by USP15-mediated cGAS activation may be a novel regulatory mechanism in the fine-tuning of innate immunity.

Blocking cGAS/STING signaling protects against sepsis-associated acute liver injury

Sepsis-associated acute liver injury (ALI) contributes to the pathogenesis of multiple organ dysfunction syndrome and thus increases mortality. Nevertheless, specific therapeutics for sepsis-associated ALI are scant so far. The cyclic GMP-AMP synthase (cGAS), a cytosolic DNA sensor, is implicated in a series of inflammatory diseases. However, whether cGAS functions in the pathogenesis of ALI is still unclear. We herein investigated the role of cGAS in the development of ALI, and if any, by which mechanism cGAS is involved. After a challenge using cecum ligation and puncture (CLP) or lipopolysaccharide (LPS) plus d-galactosamine (GalN) in WT and gene-modified mice, we found that cGAS signaling was activated, and cGAS deficiency significantly attenuated CLP- or LPS/GalN-induced liver injury, liver dysfunction and so-caused mice death. In addition, CLP or LPS/GalN augmented type I interferon signaling—the downstream of cGAS pathway. Recombinant interferon-β (rIFNβ) enhanced LPS/GalN-induced hepatocyte death and partly reversed the protection induced by cGAS depletion. Besides of inflammation, cGAS deletion was capable of preventing LPS/GalN-induced hepatocyte death. Hepatocyte-specific deletion of STING, the downstream of cGAS activation, showed a significant protection against ALI, which could be phenocopied by pharmacological inhibition of cGAS or STING via RU.521 or H-151 respectively. Taken together, cGAS/STING signaling promotes ALI by both type I IFN responses and hepatocyte death. Inhibition of cGAS/STING signaling would be a promising strategy for preventing ALI in sepsis.

The E3 ubiquitin ligase ARIH1 promotes antiviral immunity and autoimmunity by inducing mono-ISGylation and oligomerization of cGAS

The cytosolic DNA sensor cyclic GMP-AMP synthase (cGAS) plays a critical role in antiviral immunity and autoimmunity. The activity and stability of cGAS are fine-tuned by post-translational modifications. Here, we show that ariadne RBR E3 ubiquitin protein ligase 1 (ARIH1) catalyzes the mono-ISGylation and induces the oligomerization of cGAS, thereby promoting antiviral immunity and autoimmunity. Knockdown or knockout of ARIH1 significantly inhibits herpes simplex virus 1 (HSV-1)- or cytoplasmic DNA-induced expression of type I interferons (IFNs) and proinflammatory cytokines. Consistently, tamoxifen-treated ER-Cre;Arih1fl/fl mice and Lyz2-Cre; Arih1fl/fl mice are hypersensitive to HSV-1 infection compared with the controls. In addition, deletion of ARIH1 in myeloid cells alleviates the autoimmune phenotypes and completely rescues the autoimmune lethality caused by TREX1 deficiency. Mechanistically, HSV-1- or cytosolic DNA-induced oligomerization and activation of cGAS are potentiated by ISGylation at its K187 residue, which is catalyzed by ARIH1. Our findings thus reveal an important role of ARIH1 in innate antiviral and autoimmune responses and provide insight into the post-translational regulation of cGAS.

Ambient PM2.5 exposure causes cellular senescence via DNA damage, micronuclei formation, and cGAS activation

Ambient PM2.5 is one of the environmental risk factors and was correlated with senescence-related diseases based on the epidemiologic investigation. However, little is known about senescence induced by PM2.5 as well as the underlying mechanisms. In this study, we demonstrated that PM2.5 exposure aggravated cellular senescence in vivo and in vitro, and disrupted micronuclei (MN) played a vital role in this process. Our results suggested that the nuclear envelope (NE) of PM2.5-induced MN was ruptured. Subsequently, cGAS was found to localize to approximately 80% of the disrupted MN but few for intact MN. Upon examination of cGAMP and SA-β-Gal, the cGAS-STING pathway was found activated and related to cellular senescence induced by PM2.5. Taken together, we reported a novel finding that PM2.5 exposure causes cellular senescence via DNA damage, MN formation, and cGAS activation. These results revealed the potential toxicity of PM2.5 and its related mechanisms in cellular senescence.

Regulation of cGAS Activity and Downstream Signaling.

Cyclic GMP-AMP synthase (cGAS) is a predominant and ubiquitously expressed cytosolic onfirmedDNA sensor that activates innate immune responses by producing a second messenger, cyclic GMP-AMP (cGAMP), and the stimulator of interferon genes (STING). cGAS contains a highly disordered N-terminus, which can sense genomic/chromatin DNA, while the C terminal of cGAS binds dsDNA liberated from various sources, including mitochondria, pathogens, and dead cells. Furthermore, cGAS cellular localization dictates its response to foreign versus self-DNA. Recent evidence has also highlighted the importance of dsDNA-induced post-translational modifications of cGAS in modulating inflammatory responses. This review summarizes and analyzes cGAS activity regulation based on structure, sub-cellular localization, post-translational mechanisms, and Ca2+ signaling. We also discussed the role of cGAS activation in different diseases and clinical outcomes.