Neadaptivní imunitní systém je první obranou organismu při jeho napadení patogenem. Signalizační cesta cGAS -cGAMP -STING je součástí tohoto systému a je zcela klíčová při obraně proti DNA virům a retrovirům Zároveň je chronické vybuzení této kaskády pravděpodobnou příčinou některých autoimunitních onemocnění. Studium této signalizační cesty je klíčové pro vývoj terapeutik virových a autoimunitních onemocnění.
The innate immune system is the first line of defence of organism against pathogens. As a part of innate immune system, cGAS -cGAMP- -STING signaling pathway takes a crucial part in immune response to DNA viruses and retroviruses. In contrast, chronic activation of this pathway is probably cause of some autoimmune diseases. Studium of this signaling pathway is crucial for new antiviral and immunosupresive drugs development.
- Keywords
- cGAS-cGAMP-STING,
- MeSH
- DNA Viruses * immunology MeSH
- DNA genetics chemistry immunology MeSH
- Phosphorylation MeSH
- Interferon Type I * metabolism MeSH
- Humans MeSH
- Immunity, Innate * MeSH
- Receptor, Interferon alpha-beta * MeSH
- Receptors, Pattern Recognition MeSH
- Retroviridae * immunology MeSH
- Signal Transduction * MeSH
- Ubiquitins metabolism MeSH
- Check Tag
- Humans MeSH
- Publication type
- Research Support, Non-U.S. Gov't MeSH
To explore the mechanism whereby cGAS-STING pathway regulates the pyroptosis of cryptorchidism cells, with a view to finding a new strategy for clinically treating cryptorchidism-induced infertility. Spermatogonial GC-1 cells were heat stimulated to simulate the heat hurt microenvironment of cryptorchidism. The cell viability was assayed by CCK-8, and cellular DNA damage was detected by gamma-H2AX immunofluo-rescence assay. Flow cytometry was employed to assess pyroptosis index, while western blot, ELISA and PCR were used to examine the expressions of pyroptosis-related proteins (Caspase-1, IL-1beta, NLRP3) and cGAS-STING pathway proteins (cGAS, STING). After STING silencing by siRNA, the expressions of pyroptosis-related proteins were determined. Pyroptosis occurred after heat stimulation of cells. Morphological detection found cell swelling and karyopyknosis. According to the gamma-H2AX immunofluorescence (IFA) assay, the endonuclear green fluorescence was significantly enhanced, the gamma-H2AX content markedly increased, and the endonuclear DNA was damaged. Flow cytometry revealed a significant increase in pyroptosis index. Western blot and PCR assays showed that the expressions of intracellular pyrogenic proteins like Caspase-1, NLRP3 and GSDMD were elevated. The increased STING protein and gene expressions in cGAS-STING pathway suggested that the pathway was intracellularly activated. Silencing STING protein in cGAS-STING pathway led to significantly inhibited pyroptosis. These results indicate that cGAS-STING pathway plays an important role in heat stress-induced pyroptosis of spermatogonial cells. After heat stimulation of spermatogonial GC-1 cells, pyroptosis was induced and cGAS-STING pathway was activated. This study can further enrich and improve the molecular mechanism of cryptorchidism.
- MeSH
- Acetates * MeSH
- Chromogranin A MeSH
- Phenols * MeSH
- Caspase 1 MeSH
- Cryptorchidism * MeSH
- Humans MeSH
- Nucleotidyltransferases MeSH
- NLR Family, Pyrin Domain-Containing 3 Protein MeSH
- Pyroptosis MeSH
- Signal Transduction MeSH
- Spermatogonia MeSH
- Heat Stroke * MeSH
- Check Tag
- Humans MeSH
- Male MeSH
- Publication type
- Journal Article MeSH
BK polyomavirus (BKPyV) infection in humans is usually asymptomatic but ultimately results in viral persistence. In immunocompromised hosts, virus reactivation can lead to nephropathy or hemorrhagic cystitis. The urinary tract serves as a silent reservoir for the virus. Recently, it has been demonstrated that human bladder microvascular endothelial cells (HBMVECs) serve as viral reservoirs, given their unique response to infection, which involves interferon (IFN) production. The aim of the present study was to better understand the life cycle of BKPyV in HBMVECs, uncover the molecular pathway leading to IFN production, and to identify the connection between the viral life cycle and the activation of the IFN response. Here, in the early stage of infection, BKPyV virions were found in internalized monopinocytic vesicles, while later they were detected in late endosomes, lysosomes, tubuloreticular structures, and vacuole-like vesicles. The production of viral progeny in these cells started at 36 h postinfection. Increased cell membrane permeability and peaks of virion release coincided with the leakage of viral and cellular DNA into the cytosol at approximately 60 h postinfection. Leaked DNA colocalized with and activated cGAS, leading to the activation of STING and the consequent transcription of IFNB and IFN-related genes; in contrast, the IFN response was attenuated by exposure to the cGAS inhibitor, G140. These findings highlight the importance of the cGAS-STING pathway in the innate immune response of HBMVECs to BKPyV.
- MeSH
- Endothelial Cells * virology MeSH
- Interferons metabolism MeSH
- Cells, Cultured MeSH
- Humans MeSH
- Membrane Proteins metabolism genetics MeSH
- Urinary Bladder * virology MeSH
- Nucleotidyltransferases metabolism genetics MeSH
- Polyomavirus Infections virology immunology MeSH
- Virus Replication MeSH
- Signal Transduction * MeSH
- Virion MeSH
- BK Virus * physiology MeSH
- Check Tag
- Humans MeSH
- Publication type
- Journal Article MeSH
OBJECTIVE: The TLR3/cGAS-STING-IFN signaling has recently been reported to be disturbed in colorectal cancer due to deregulated expression of the genes involved. Our study aimed to investigate the influence of potential regulatory variants in these genes on the risk of sporadic colorectal cancer (CRC) in a Czech cohort of 1424 CRC patients and 1114 healthy controls. METHODS: The variants in the TLR3, CGAS, TMEM173, IKBKE, and TBK1 genes were selected using various online bioinformatic tools, such as UCSC browser, HaploReg, Regulome DB, Gtex Portal, SIFT, PolyPhen2, and miRNA prediction tools. RESULTS: Logistic regression analysis adjusted for age and sex detected a nominal association between CRC risk and three variants, CGAS rs72960018 (OR: 1.68, 95% CI: 1.11-2.53, P-value = .01), CGAS rs9352000 (OR: 2.02, 95% CI: 1.07-3.84, P-value = .03) and TMEM173 rs13153461 (OR: 1.53, 95% CI: 1.03-2.27, P-value = .03). Their cumulative effect revealed a threefold increased CRC risk in carriers of 5-6 risk alleles compared to those with 0-2 risk alleles. Epistatic interactions between these genes and the previously genotyped IFNAR1, IFNAR2, IFNA, IFNB, IFNK, IFNW, IRF3, and IRF7 genes, were computed to test their effect on CRC risk. Overall, we obtained nine pair-wise interactions within and between the CGAS, TMEM173, IKBKE, and TBK1 genes. Two of them remained statistically significant after Bonferroni correction. Additional 52 interactions were observed when IFN variants were added to the analysis. CONCLUSIONS: Our data suggest that epistatic interactions and a high number of risk alleles may play an important role in CRC carcinogenesis, offering novel biological understanding for the CRC management.
- MeSH
- Adult MeSH
- Epistasis, Genetic * MeSH
- Genotyping Techniques MeSH
- Interferons genetics MeSH
- Polymorphism, Single Nucleotide MeSH
- Carcinogenesis genetics MeSH
- I-kappa B Kinase genetics MeSH
- Cohort Studies MeSH
- Colon diagnostic imaging pathology MeSH
- Colonoscopy MeSH
- Colorectal Neoplasms diagnosis genetics pathology MeSH
- Middle Aged MeSH
- Humans MeSH
- Membrane Proteins genetics MeSH
- Adolescent MeSH
- Young Adult MeSH
- Nucleotidyltransferases genetics MeSH
- Protein Serine-Threonine Kinases genetics MeSH
- Gene Expression Regulation, Neoplastic * MeSH
- Rectum diagnostic imaging pathology MeSH
- Aged, 80 and over MeSH
- Aged MeSH
- Signal Transduction genetics MeSH
- Case-Control Studies MeSH
- Toll-Like Receptor 3 genetics MeSH
- Computational Biology MeSH
- Healthy Volunteers MeSH
- Check Tag
- Adult MeSH
- Middle Aged MeSH
- Humans MeSH
- Adolescent MeSH
- Young Adult MeSH
- Male MeSH
- Aged, 80 and over MeSH
- Aged MeSH
- Female MeSH
- Publication type
- Journal Article MeSH
- Observational Study MeSH
- Research Support, Non-U.S. Gov't MeSH
The cGAS-STING (cyclic GMP-AMP synthase-stimulator of interferon genes) pathway plays a crucial role in inducing an antiviral and antitumor immune response. We studied the effects of synthetic STING agonists on several immune populations and related cytokine production. In comparison with the toll-like receptor 7 (TLR7) agonist, STING agonists induced secretion of a broader proinflammatory cytokine spectrum. Unlike the TLR7 agonist, the structurally diverse STING agonists partially depleted B and NK cells and completely depleted CD14+ monocytes via induction of apoptosis. The TANK-binding kinase 1 inhibitor efficiently prevented interferon alpha (IFNα) secretion and cell depletion, suggesting their possible dependence on the cGAS-STING pathway activation. Finally, IFNα, tumor necrosis factor alpha, interleukin 6, and interleukin 1 beta secretion and CD14+ monocyte apoptosis were primary responses to STING agonists, whereas IFNγ was secreted secondarily. These findings bring new insights into the cGAS-STING pathway immunomodulation that is of future therapeutic importance.
Sensing of cytoplasmic DNA by cyclic guanosine monophosphate-adenosine monophosphate (cGAMP) synthase (cGAS) results in production of the dinucleotide cGAMP and consecutive activation of stimulator of interferon genes (STING) followed by production of type I interferon (IFN). Although cancer cells contain supra-normal concentrations of cytoplasmic DNA, they rarely produce type I IFN spontaneously. This suggests that defects in the DNA-sensing pathway may serve as an immune escape mechanism. We find that cancer cells produce cGAMP that is transferred via gap junctions to tumor-associated dendritic cells (DCs) and macrophages, which respond by producing type I IFN in situ. Cancer-cell-intrinsic expression of cGAS, but not STING, promotes infiltration by effector CD8+ T cells and consequently results in prolonged survival. Furthermore, cGAS-expressing cancers respond better to genotoxic treatments and immunotherapy. Thus, cancer-cell-derived cGAMP is crucial to protective anti-tumor CD8+ T cell immunity. Consequently, cancer-cell-intrinsic expression of cGAS determines tumor immunogenicity and makes tumors hot. These findings are relevant for genotoxic and immune therapies for cancer.
- MeSH
- CD8-Positive T-Lymphocytes immunology MeSH
- Dendritic Cells metabolism MeSH
- Immunotherapy MeSH
- Interferon Type I metabolism MeSH
- Humans MeSH
- Membrane Proteins MeSH
- Microsatellite Repeats genetics MeSH
- Mice, Inbred C57BL MeSH
- Cell Line, Tumor MeSH
- Neoplasms drug therapy immunology pathology MeSH
- Nucleotides, Cyclic metabolism MeSH
- Nucleotidyltransferases metabolism MeSH
- DNA Damage MeSH
- Disease Progression MeSH
- Animals MeSH
- Check Tag
- Humans MeSH
- Animals MeSH
- Publication type
- Journal Article MeSH
- Research Support, Non-U.S. Gov't MeSH
The mechanism by which DNA viruses interact with different DNA sensors and their connection with the activation of interferon (IFN) type I pathway are poorly understood. We investigated the roles of protein 204 (p204) and cyclic guanosine-adenosine synthetase (cGAS) sensors during infection with mouse polyomavirus (MPyV). The phosphorylation of IFN regulatory factor 3 (IRF3) and the stimulator of IFN genes (STING) proteins and the upregulation of IFN beta (IFN-β) and MX Dynamin Like GTPase 1 (MX-1) genes were detected at the time of replication of MPyV genomes in the nucleus. STING knockout abolished the IFN response. Infection with a mutant virus that exhibits defective nuclear entry via nucleopores and that accumulates in the cytoplasm confirmed that replication of viral genomes in the nucleus is required for IFN induction. The importance of both DNA sensors, p204 and cGAS, in MPyV-induced IFN response was demonstrated by downregulation of the IFN pathway observed in p204-knockdown and cGAS-knockout cells. Confocal microscopy revealed the colocalization of p204 with MPyV genomes in the nucleus. cGAS was found in the cytoplasm, colocalizing with viral DNA leaked from the nucleus and with DNA within micronucleus-like bodies, but also with the MPyV genomes in the nucleus. However, 2'3'-Cyclic guanosine monophosphate-adenosine monophosphate synthesized by cGAS was detected exclusively in the cytoplasm. Biochemical assays revealed no evidence of functional interaction between cGAS and p204 in the nucleus. Our results provide evidence for the complex interactions of MPyV and DNA sensors including the sensing of viral genomes in the nucleus by p204 and of leaked viral DNA and micronucleus-like bodies in the cytoplasm by cGAS.
- MeSH
- DNA, Viral genetics immunology MeSH
- Phosphoproteins antagonists & inhibitors genetics metabolism MeSH
- Phosphorylation MeSH
- Tumor Virus Infections immunology virology MeSH
- Host-Pathogen Interactions MeSH
- Interferon-beta metabolism MeSH
- Nuclear Proteins antagonists & inhibitors genetics metabolism MeSH
- Membrane Proteins antagonists & inhibitors genetics metabolism MeSH
- Mice MeSH
- Nucleotidyltransferases antagonists & inhibitors genetics metabolism MeSH
- Polyomavirus Infections immunology virology MeSH
- Polyomavirus genetics immunology MeSH
- Immunity, Innate immunology MeSH
- Animals MeSH
- Check Tag
- Mice MeSH
- Animals MeSH
- Publication type
- Journal Article MeSH
- Research Support, Non-U.S. Gov't MeSH
Cyclic dinucleotides are second messengers in the cyclic GMP-AMP synthase (cGAS)-stimulator of interferon genes (STING) pathway, which plays an important role in recognizing tumor cells and viral or bacterial infections. They bind to the STING adaptor protein and trigger expression of cytokines via TANK binding kinase 1 (TBK1)/interferon regulatory factor 3 (IRF3) and inhibitor of nuclear factor-κB (IκB) kinase (IKK)/nuclear factor-κB (NFκB) signaling cascades. In this work, we describe an enzymatic preparation of 2'-5',3'-5'-cyclic dinucleotides (2'3'CDNs) with use of cyclic GMP-AMP synthases (cGAS) from human, mouse, and chicken. We profile substrate specificity of these enzymes by employing a small library of nucleotide-5'-triphosphate (NTP) analogues and use them to prepare 33 2'3'CDNs. We also determine affinity of these CDNs to five different STING haplotypes in cell-based and biochemical assays and describe properties needed for their optimal activity toward all STING haplotypes. Next, we study their effect on cytokine and chemokine induction by human peripheral blood mononuclear cells (PBMCs) and evaluate their cytotoxic effect on monocytes. Additionally, we report X-ray crystal structures of two new CDNs bound to STING protein and discuss structure-activity relationship by using quantum and molecular mechanical (QM/MM) computational modeling.
- MeSH
- Biological Assay MeSH
- Cytokines metabolism MeSH
- HEK293 Cells MeSH
- Protein Conformation MeSH
- Leukocytes, Mononuclear drug effects MeSH
- Humans MeSH
- Membrane Proteins chemistry metabolism MeSH
- Nucleotides, Cyclic chemical synthesis pharmacology MeSH
- Computer Simulation MeSH
- Gene Expression Regulation drug effects MeSH
- Structure-Activity Relationship MeSH
- Check Tag
- Humans MeSH
- Publication type
- Journal Article MeSH
- Research Support, Non-U.S. Gov't MeSH
Cyclic dinucleotides (CDNs) are second messengers that activate stimulator of interferon genes (STING). The cGAS-STING pathway plays a promising role in cancer immunotherapy. Here, we describe the synthesis of CDNs containing 7-substituted 7-deazapurine moiety. We used mouse cyclic GMP-AMP synthase and bacterial dinucleotide synthases for the enzymatic synthesis of CDNs. Alternatively, 7-(het)aryl 7-deazapurine CDNs were prepared by Suzuki-Miyaura cross-couplings. New CDNs were tested in biochemical and cell-based assays for their affinity to human STING. Eight CDNs showed better activity than 2'3'-cGAMP, the natural ligand of STING. The effect on cytokine and chemokine induction was also evaluated. The best activities were observed for CDNs bearing large aromatic substituents that point above the CDN molecule. We solved four X-ray structures of complexes of new CDNs with human STING. We observed π-π stacking interactions between the aromatic substituents and Tyr240 that are involved in the stabilization of CDN-STING complexes.
- MeSH
- Cytokines MeSH
- Interferons MeSH
- Humans MeSH
- Ligands MeSH
- Membrane Proteins * metabolism MeSH
- Mice MeSH
- Nucleotides, Cyclic * chemistry MeSH
- Nucleotidyltransferases MeSH
- Animals MeSH
- Check Tag
- Humans MeSH
- Mice MeSH
- Animals MeSH
- Publication type
- Journal Article MeSH
- Research Support, Non-U.S. Gov't MeSH
Cyclic dinucleotides (CDNs) trigger the cyclic GMP-AMP synthase-stimulator of interferon genes (cGAS-STING) pathway, which plays a key role in cytosolic DNA sensing and thus in immunomodulation against infections, cell damage and cancer. However, cancer immunotherapy trials with CDNs have shown immune activation, but not complete tumor regression. Nevertheless, we designed a novel class of CDNs containing vinylphosphonate based on a STING-affinity screening assay. In vitro, acyloxymethyl phosphate/phosphonate prodrugs of these vinylphosphonate CDNs were up to 1000-fold more potent than the clinical candidate ADU-S100. In vivo, the lead prodrug induced tumor-specific T cell priming and facilitated tumor regression in the 4T1 syngeneic mouse model of breast cancer. Moreover, we solved the crystal structure of this ligand bound to the STING protein. Therefore, our findings not only validate the therapeutic potential of vinylphosphonate CDNs but also open up opportunities for drug development in cancer immunotherapy bridging innate and adaptive immunity.
- MeSH
- DNA MeSH
- Immunotherapy MeSH
- Mice MeSH
- Neoplasms * drug therapy MeSH
- Nucleotides, Cyclic * pharmacology metabolism MeSH
- Immunity, Innate MeSH
- Animals MeSH
- Check Tag
- Mice MeSH
- Animals MeSH
- Publication type
- Journal Article MeSH
