Interplay of cGAS with micronuclei: Regulation and diseases.

In higher eukaryotes, sophisticate regulation of genome function requires all chromosomes to be packed into a single nucleus. Micronucleus (MN), the dissociative nucleus-like structure frequently observed in aging and multiple disease settings, has critical, yet under-recognized, pathophysiological functions. Micronuclei (MNi) have recently emerged as major sources of cytosolic DNA that can activate the cGAS-STING axis in a cell-intrinsic manner. However, MNi induced from different genotoxic stressors display great heterogeneity in binding or activating cGAS and the signaling responses downstream of the MN-induced cGAS-STING axis have divergent outcomes including autoimmunity, autoinflammation, metastasis, or cell death. Thus, full characterization of molecular network underpinning the interplay of cGAS and MN is important to elucidate the pathophysiological roles of immunogenic MN and design improved drugs that selectively target cancer via boosting the MN-derived cGAS-STING axis. Here, we summarize our current understanding of the mechanisms for self-DNA discrimination by cGAS. We focus on discussing how MN immunogencity is dictated by multiple mechanisms including integrity of micronuclear envelope, state of nucleosome and DNA, competitive factors, damaged mitochondrial DNA and micronucleophagy. We also describe emerging links between immunogenic MN and human diseases including cancer, neurodegenerative diseases and COVID-19. Particularly, we explore the exciting concept of inducing immunogenic MN as a therapeutic approach in treating cancer. We propose a new theoretical framework to describe immunogenic MN as a biological sensor to modulate cellular processes in response to genotoxic stress and provide perspectives on developing novel experimental approaches to unravel the complexity of MN immunogenicity regulation and immunogenic MN pathophysiology.

Phase-separated nucleocapsid protein of SARS-CoV-2 suppresses cGAS-DNA recognition by disrupting cGAS-G3BP1 complex.

Currently, the incidence and fatality rate of SARS-CoV-2 remain continually high worldwide. COVID-19 patients infected with SARS-CoV-2 exhibited decreased type I interferon (IFN-I) signal, along with limited activation of antiviral immune responses as well as enhanced viral infectivity. Dramatic progresses have been made in revealing the multiple strategies employed by SARS-CoV-2 in impairing canonical RNA sensing pathways. However, it remains to be determined about the SARS-CoV-2 antagonism of cGAS-mediated activation of IFN responses during infection. In the current study, we figure out that SARS-CoV-2 infection leads to the accumulation of released mitochondria DNA (mtDNA), which in turn triggers cGAS to activate IFN-I signaling. As countermeasures, SARS-CoV-2 nucleocapsid (N) protein restricts the DNA recognition capacity of cGAS to impair cGAS-induced IFN-I signaling. Mechanically, N protein disrupts the assembly of cGAS with its co-factor G3BP1 by undergoing DNA-induced liquid-liquid phase separation (LLPS), subsequently impairs the double-strand DNA (dsDNA) detection ability of cGAS. Taken together, our findings unravel a novel antagonistic strategy by which SARS-CoV-2 reduces DNA-triggered IFN-I pathway through interfering with cGAS-DNA phase separation.

NETs Promote Inflammatory Injury by Activating cGAS-STING Pathway in Acute Lung Injury.

Acute respiratory distress syndrome (ARDS) threatens the survival of critically ill patients, the mechanisms of which are still unclear. Neutrophil extracellular traps (NETs) released by activated neutrophils play a critical role in inflammatory injury. We investigated the role of NETs and the underlying mechanism involved in acute lung injury (ALI). We found a higher expression of NETs and cyclic GMP-AMP synthase-stimulator of interferon genes (cGAS-STING) in the airways, which was reduced by Deoxyribonuclease I (DNase I) in ALI. The administration of the STING inhibitor H-151 also significantly relieved inflammatory lung injury, but failed to affect the high expression of NETs in ALI. We isolated murine neutrophils from bone marrow and acquired human neutrophils by inducing HL-60 to differentiate. After the PMA interventions, exogenous NETs were obtained from such extracted neutrophils. Exogenous NETs intervention in vitro and in vivo resulted in airway injury, and such inflammatory lung injury was reversed upon degrading NETs with or inhibiting cGAS-STING with H-151 as well as siRNA STING. In conclusion, cGAS-STING participates in regulating NETs-mediated inflammatory pulmonary injury, which is expected to be a new therapeutic target for ARDS/ALI.

Recent advances in recombinase polymerase amplification: Principle, advantages, disadvantages and applications.

After the outbreak of SARS-CoV-2, nucleic acid testing quickly entered people's lives. In addition to the polymerase chain reaction (PCR) which was commonly used in nucleic acid testing, isothermal amplification methods were also important nucleic acid testing methods. Among several common isothermal amplification methods like displaced amplification, rolling circle amplification, and so on, recombinase polymerase amplification (RPA) was recently paid more attention to. It had the advantages like a simple operation, fast amplification speed, and reaction at 37-42°C, et al. So it was very suitable for field detection. However, there were still some disadvantages to RPA. Herein, our review mainly summarized the principle, advantages, and disadvantages of RPA. The specific applications of RPA in bacterial detection, fungi detection, virus detection, parasite detection, drug resistance gene detection, genetically modified food detection, and SARS-CoV-2 detection were also described. It was hoped that the latest research progress on RPA could be better delivered to the readers who were interested in RPA.

Emerging role of the cGAS-STING signaling pathway in autoimmune diseases: Biologic function, mechanisms and clinical prospection.

The cyclic GMP-AMP synthase-stimulator of interferon genes (cGAS-STING) signaling pathway, as vital component of innate immune system, acts a vital role in distinguishing invasive pathogens and cytosolic DNA. Cytosolic DNA sensor cGAS first binds to cytosolic DNA and catalyzes synthesis of cyclic guanosine monophosphate-adenosine monophosphate (cGAMP), which is known as the second messenger. Next, cGAMP activates the adaptor protein STING, triggering a molecular chain reaction to stimulate cytokines including interferons (IFNs). Recently, many researches have revealed that the regulatory role of cGAS-STING signaling pathway in autoimmune diseases (AIDs) such as Rheumatoid arthritis (RA), Aicardi Goutières syndrome (AGS) and systemic lupus erythematosus (SLE). Moreover, accumulated evidence have showed inhibition of the cGAS-STING signaling pathway could remarkably suppress the joint swelling and inflammatory cell infiltration in RA mice. Therefore, in this review, we describe the molecular properties, biologic function and mechanisms of the cGAS-STING signaling pathway in AIDs. In addition, potential clinical applications especially selective small molecule inhibitors targeting the cGAS-STING signaling pathway are also discussed.

SARS-CoV-2 ORF10 antagonizes STING-dependent interferon activation and autophagy.

A characteristic feature of COVID-19, the disease caused by severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) infection, is the dysregulated immune response with impaired type I and III interferon (IFN) expression and an overwhelming inflammatory cytokine storm. RIG-I-like receptors (RLRs) and cGAS-STING signaling pathways are responsible for sensing viral infection and inducing IFN production to combat invading viruses. Multiple proteins of SARS-CoV-2 have been reported to modulate the RLR signaling pathways to achieve immune evasion. Although SARS-CoV-2 infection also activates the cGAS-STING signaling by stimulating micronuclei formation during the process of syncytia, whether SARS-CoV-2 modulates the cGAS-STING pathway requires further investigation. Here, we screened 29 SARS-CoV-2-encoded viral proteins to explore the viral proteins that affect the cGAS-STING signaling pathway and found that SARS-CoV-2 open reading frame 10 (ORF10) targets STING to antagonize IFN activation. Overexpression of ORF10 inhibits cGAS-STING-induced interferon regulatory factor 3 phosphorylation, translocation, and subsequent IFN induction. Mechanistically, ORF10 interacts with STING, attenuates the STING-TBK1 association, and impairs STING oligomerization and aggregation and STING-mediated autophagy; ORF10 also prevents the endoplasmic reticulum (ER)-to-Golgi trafficking of STING by anchoring STING in the ER. Taken together, these findings suggest that SARS-CoV-2 ORF10 impairs the cGAS-STING signaling by blocking the translocation of STING and the interaction between STING and TBK1 to antagonize innate antiviral immunity.

SARS-CoV-2 spike protein-induced cell fusion activates the cGAS-STING pathway and the interferon response.

Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) caused the unprecedented coronavirus disease 2019 (COVID-19) pandemic. Critical cases of COVID-19 are characterized by the production of excessive amounts of cytokines and extensive lung damage, which is partially caused by the fusion of SARS-CoV-2-infected pneumocytes. Here, we found that cell fusion caused by the SARS-CoV-2 spike (S) protein induced a type I interferon (IFN) response. This function of the S protein required its cleavage by proteases at the S1/S2 and the S2' sites. We further showed that cell fusion damaged nuclei and resulted in the formation of micronuclei that were sensed by the cytosolic DNA sensor cGAS and led to the activation of its downstream effector STING. Phosphorylation of the transcriptional regulator IRF3 and the expression of IFNB, which encodes a type I IFN, were abrogated in cGAS-deficient fused cells. Moreover, infection with VSV-SARS-CoV-2 also induced cell fusion, DNA damage, and cGAS-STING-dependent expression of IFNB. Together, these results uncover a pathway underlying the IFN response to SARS-CoV-2 infection. Our data suggest a mechanism by which fused pneumocytes in the lungs of patients with COVID-19 may enhance the production of IFNs and other cytokines, thus exacerbating disease severity.

STING agonist diABZI induces PANoptosis and DNA mediated acute respiratory distress syndrome (ARDS).

Stimulator of interferon genes (STING) contributes to immune responses against tumors and may control viral infection including SARS-CoV-2 infection. However, activation of the STING pathway by airway silica or smoke exposure leads to cell death, self-dsDNA release, and STING/type I IFN dependent acute lung inflammation/ARDS. The inflammatory response induced by a synthetic non-nucleotide-based diABZI STING agonist, in comparison to the natural cyclic dinucleotide cGAMP, is unknown. A low dose of diABZI (1 µg by endotracheal route for 3 consecutive days) triggered an acute neutrophilic inflammation, disruption of the respiratory barrier, DNA release with NET formation, PANoptosis cell death, and inflammatory cytokines with type I IFN dependent acute lung inflammation. Downstream upregulation of DNA sensors including cGAS, DDX41, IFI204, as well as NLRP3 and AIM2 inflammasomes, suggested a secondary inflammatory response to dsDNA as a danger signal. DNase I treatment, inhibition of NET formation together with an investigation in gene-deficient mice highlighted extracellular DNA and TLR9, but not cGAS, as central to diABZI-induced neutrophilic response. Therefore, activation of acute cell death with DNA release may lead to ARDS which may be modeled by diABZI. These results show that airway targeting by STING activator as a therapeutic strategy for infection may enhance lung inflammation with severe ARDS. STING agonist diABZI induces neutrophilic lung inflammation and PANoptosis A, Airway STING priming induce a neutrophilic lung inflammation with epithelial barrier damage, double-stranded DNA release in the bronchoalvelolar space, cell death, NETosis and type I interferon release. B, 1. The diamidobenzimidazole (diABZI), a STING agonist is internalized into the cytoplasm through unknown receptor and induce the activation and dimerization of STING followed by TBK1/IRF3 phosporylation leading to type I IFN response. STING activation also leads to NF-kB activation and the production of pro-inflammatory cytokines TNFα and IL-6. 2. The activation of TNFR1 and IFNAR1 signaling pathway results in ZBP1 and RIPK3/ASC/CASP8 activation leading to MLKL phosphorylation and necroptosis induction. 3. This can also leads to Caspase-3 cleavage and apoptosis induction. 4. Self-dsDNA or mtDNA sensing by NLRP3 or AIM2 induces inflammsome formation leading to Gasdermin D cleavage enabling Gasdermin D pore formation and the release mature IL-1ß and pyroptosis. NLRP3 inflammasome formation can be enhanced by the ZBP1/RIPK3/CASP8 complex. 5. A second signal of STING activation with diABZI induces cell death and the release of self-DNA which is sensed by cGAS and form 2'3'-cGAMP leading to STING hyper activation, the amplification of TBK1/IRF3 and NF-kB pathway and the subsequent production of IFN-I and inflammatory TNFα and IL-6. This also leads to IFI204 and DDX41 upregulation thus, amplifying the inflammatory loop. The upregulation of apoptosis, pyroptosis and necroptosis is indicative of STING-dependent PANoptosis.

Dysfunctional telomeres through mitostress-induced cGAS/STING activation to aggravate immune senescence and viral pneumonia.

Disproportionately high incidence and mortality of respiratory infection such as influenza A virus (IAV) and SARS-CoV-2 have been evidenced in the elderly, but the role and the mechanism of age-associated immune deregulation in disease exacerbation are not well defined. Using a late generation of mice deficient in telomerase RNA (Terc-/- ), we herein demonstrated that aged mice were exquisitely susceptible to respiratory viral infection, with excessive inflammation and increased mortality. Furthermore, we identified the cGAS/STING pathway, which was essentially induced by the leaked mitochondrial DNA, as a biologically relevant mechanism contributing to exaggerated inflammation in Terc-/- mice following viral infection. Innate immune cells, mainly, macrophages with shortened telomeres, exhibited hallmarks of cellular senescence, mitochondrial distress, and aberrant activation of STING and NLRP3 inflammasome pathways, which predisposed mice to severe viral pneumonia during commonly mild infections. Application of STING inhibitor and, more importantly, senolytic agent, reduced the burden of stressed macrophages, improved mitochondrial integrity, and suppressed STING activation, thereby conferring the protection for Terc-/- mice against respiratory infection. Together, the findings expand our understanding of innate immune senescence and reveal the potential of the senolytics as a promising treatment to alleviate the symptom of viral pneumonia, particularly for the older population.

Crosstalk between RNA viruses and DNA sensors: Role of the cGAS-STING signalling pathway.

Despite only comprising half of all known viral species, RNA viruses are disproportionately responsible for many of the worst epidemics in human history, including outbreaks of influenza, poliomyelitis, Ebola, and most recently, the coronavirus disease-2019 (COVID-19) pandemic. The propensity for RNA viruses to replicate in cytosolic compartments has led to an evolutionary arms race and the emergence of cytosolic sensors to recognise and initiate the host innate immune response. Although significant progress has been made in identifying and characterising cytosolic RNA sensors as anti-viral innate immune factors, the potential role for cytosolic DNA sensors in RNA viral infection is only recently being appreciated. Among these, the cyclic GMP-AMP synthase (cGAS)-stimulator of interferon genes (STING) pathway has attracted increasing attention. The cGAS-STING signalling pathway has emerged as a key innate immune signalling axis that is implicated in diverse human diseases from infectious diseases to neurodegeneration and cancer. Here we review the existing literature on RNA viruses and their reciprocal interactions with the cGAS-STING pathway and share insights into RNA virus diversity by touching on the similarities and differences of RNA viral strategies.

Sensing of cytoplasmic chromatin by cGAS activates innate immune response in SARS-CoV-2 infection.

The global coronavirus disease 2019 (COVID-19) pandemic is caused by severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), a positive-sense RNA virus. How the host immune system senses and responds to SARS-CoV-2 infection remain largely unresolved. Here, we report that SARS-CoV-2 infection activates the innate immune response through the cytosolic DNA sensing cGAS-STING pathway. SARS-CoV-2 infection induces the cellular level of 2'3'-cGAMP associated with STING activation. cGAS recognizes chromatin DNA shuttled from the nucleus as a result of cell-to-cell fusion upon SARS-CoV-2 infection. We further demonstrate that the expression of spike protein from SARS-CoV-2 and ACE2 from host cells is sufficient to trigger cytoplasmic chromatin upon cell fusion. Furthermore, cytoplasmic chromatin-cGAS-STING pathway, but not MAVS-mediated viral RNA sensing pathway, contributes to interferon and pro-inflammatory gene expression upon cell fusion. Finally, we show that cGAS is required for host antiviral responses against SARS-CoV-2, and a STING-activating compound potently inhibits viral replication. Together, our study reported a previously unappreciated mechanism by which the host innate immune system responds to SARS-CoV-2 infection, mediated by cytoplasmic chromatin from the infected cells. Targeting the cytoplasmic chromatin-cGAS-STING pathway may offer novel therapeutic opportunities in treating COVID-19. In addition, these findings extend our knowledge in host defense against viral infection by showing that host cells' self-nucleic acids can be employed as a "danger signal" to alarm the immune system.

SARS-CoV-2 ORF9b antagonizes type I and III interferons by targeting multiple components of the RIG-I/MDA-5-MAVS, TLR3-TRIF, and cGAS-STING signaling pathways.

The suppression of types I and III interferon (IFN) responses by severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) contributes to the pathogenesis of coronavirus disease 2019 (COVID-19). The strategy used by SARS-CoV-2 to evade antiviral immunity needs further investigation. Here, we reported that SARS-CoV-2 ORF9b inhibited types I and III IFN production by targeting multiple molecules of innate antiviral signaling pathways. SARS-CoV-2 ORF9b impaired the induction of types I and III IFNs by Sendai virus and poly (I:C). SARS-CoV-2 ORF9b inhibited the activation of types I and III IFNs induced by the components of cytosolic dsRNA-sensing pathways of RIG-I/MDA5-MAVS signaling, including RIG-I, MDA-5, MAVS, TBK1, and IKKε, rather than IRF3-5D, which is the active form of IRF3. SARS-CoV-2 ORF9b also suppressed the induction of types I and III IFNs by TRIF and STING, which are the adaptor protein of the endosome RNA-sensing pathway of TLR3-TRIF signaling and the adaptor protein of the cytosolic DNA-sensing pathway of cGAS-STING signaling, respectively. A mechanistic analysis revealed that the SARS-CoV-2 ORF9b protein interacted with RIG-I, MDA-5, MAVS, TRIF, STING, and TBK1 and impeded the phosphorylation and nuclear translocation of IRF3. In addition, SARS-CoV-2 ORF9b facilitated the replication of the vesicular stomatitis virus. Therefore, the results showed that SARS-CoV-2 ORF9b negatively regulates antiviral immunity and thus facilitates viral replication. This study contributes to our understanding of the molecular mechanism through which SARS-CoV-2 impairs antiviral immunity and provides an essential clue to the pathogenesis of COVID-19.

Recurrent pneumonia in a patient with new coronavirus infection after discharge from hospital for insufficient antibody production: a case report.

BACKGROUND: The rapid spread of coronavirus disease 2019 (COVID-19) was declared as an emerging public health threat by the World Health Organization. As various measures have been taken successfully to combat the epidemic caused by SARS-CoV-2, a growing number of fully recovered patients have been discharged from hospitals. However, some of them have relapsed. Little is known about the causes that triggered the relapse. CASE PRESENTATION: We report a case of a 40 years old man who suffered from recurrent pulmonary infection with progression of lesions on chest computed tomography (CT), elevated levels of ferritin and IL2R, reduced lymphocyte count and positive oropharyngeal swab test for SARS-CoV-2 again after 5 days discharge from hospital. The anti-SARS-CoV-2 antibody level of this patient was very low at the time of relapse, suggesting a weak humoral immune response to the virus. Total exon sequencing revealed mutations in TRNT1 gene, which may be responsible for B cell immunodeficiency. Therefore, uncleared SARS-CoV-2 at his first discharge was likely to lead to his recurrence. However, viral superinfection and non-infectious organizing pneumonia could not be completely excluded. CONCLUSION: COVID-19 relapse may occur in a part of discharged patients with low titers of anti-SARS-CoV-2 antibodies. These patients should be maintained in isolation for longer time even after discharge. A more sensitive method to detect SARS-CoV-2 needs to be established and serological testing for specific antibodies may be used as a reference to determine the duration of isolation.