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Aims Lifestyle-related diseases promote atherosclerosis, a chronic inflammatory disease; however, the molecular mechanism remains largely unknown. Endogenous DNA fragments released under over-nutrient condition provoke sterile inflammation through the recognition by DNA sensors. Here, we investigated the role of stimulator of interferon genes (STING), a cytosolic DNA sensor, in atherogenesis. Methods and results Apolipoprotein E-deficient (Apoe−/−) mice fed a western-type diet (WTD), a hypercholesterolaemic mouse model, showed higher STING expression and markers for DNA damage such as γH2AX, p53, and single-stranded DNA (ssDNA) accumulation in macrophages in the aorta compared with wild-type (WT) mice. The level of cGAMP, a STING agonist, in the aorta was higher in Apoe−/− mice. Genetic deletion of Sting in Apoe−/− mice reduced atherosclerotic lesions in the aortic arch, lipid, and macrophage accumulation in plaques, and inflammatory molecule expression in the aorta compared with the control. Pharmacological blockade of STING using a specific inhibitor, C-176, ameliorated atherogenesis in Apoe−/− mice. In contrast, bone marrow-specific STING expression in Apoe−/− mice stimulated atherogenesis. Expression or deletion of STING did not affect metabolic parameters and blood pressure. In vitro studies revealed that STING activation by cGAMP or mitochondrial DNA accelerated inflammatory molecule expression (e.g. TNF-α or IFN-β) in mouse and human macrophages. Activation of nuclear factor-κB and TANK binding kinase 1 was involved in STING-associated vascular inflammation and macrophage activation. Furthermore, human atherosclerotic lesions in the carotid arteries expressed STING and cGAMP. Conclusion Stimulator of interferon genes stimulates pro-inflammatory activation of macrophages, leading to the development of atherosclerosis. Stimulator of interferon genes signalling may serve as a potential therapeutic target for atherosclerosis. [ABSTRACT FROM AUTHOR]

Herpes simplex virus type 1 (HSV-1) infected cells release extracellular vesicles that deliver to uninfected cells viral factors and host components such as the stimulator of interferon genes (STING) which activates type I interferon upon foreign DNA sensing. The functions of EVs released by HSV-1 infected cells have remained unknown. Here, we describe a procedure to separate the EVs from HSV-1 virions that is based on an iodixanol/sucrose gradient. STING along with the EV markers CD63 and CD9 were found in light density fractions while HSV components accumulated in heavy density fractions. HSV-1 infection stimulated the release of EVs from the cells. The EVs derived from infected, but not from uninfected cells, activated innate immunity in recipient cells and suppressed viral gene expression and virus replication. Moreover, only the EVs derived from infected cells stimulated the expression of a subset of M1-type markers in recipient macrophages. Conversely, EVs derived from STING-knockdown cells failed to stimulate the expression of these M1-type markers, they activated innate immune responses to a lesser extent in recipient cells and they did not sustain the inhibition of virus replication. These data suggest that STING from the EV-donor cells contributes to the antiviral responses in cells receiving EVs from HSV-1 infected cells. Perturbations in the biogenesis of EVs by silencing CD63 or blocking the activity of the neutral spingomyelinase-2 (nSMase-2) increased HSV-1 yields. Overall, our data suggest that the EVs released from HSV-1 infected cells negatively impact the infection and could control the dissemination of the virus. IMPORTANCE Extracellular vesicles are released by all types of cells as they constitute major mechanism of intercellular communication and have the capacity to alter the functions of recipient cells despite their limited capacity for cargo. How the EVs released by HSV-infected cells could alter the surrounding microenvironment and influence the infection currently remains unknown. The cargo of EVs reflects the physiological state of the cells in which they were produced so the content of EVs originating from infected cells is expected to be substantially different from that of healthy cells. Our studies indicate that the EVs released by HSV-1 infected cells carry innate immune components such as STING and other host and viral factors, they can activate innate immune responses in recipient cells and inhibit HSV-1 replication. The implication of these data is that the EVs released by HSV-1 infected cells could control HSV-1 dissemination promoting its persistence in the host. [ABSTRACT FROM AUTHOR]

Stimulator of interferon genes (STING) is essential for the type I interferon and pro-inflammatory responses against DNA pathogens. In response to the presence of cytosolic DNA, STING translocates from the endoplasmic reticulum (ER) to the Golgi, and activates TANK-binding kinase 1 (TBK1), a cytosolic kinase that is essential for the activation of STING-dependent downstream signalling. The organelles where TBK1 binds to STING remain unknown. Here we show that TBK1 binds to STING at the Golgi, not at the ER. Treatment with brefeldin A, an agent to block ER-to-Golgi traffic, or knockdown of Sar1, a small GTPase that regulates coat protein complex II (COP-II)-mediated ER-to-Golgi traffic, inhibited the binding of TBK1 to STING. Endogenous TBK1 was recruited to the Golgi when STING was transported to the Golgi, as shown by immunofluorescence microscopy. STING variants that constitutively induce the type I interferon response were found in patients with autoinflammatory diseases. Even these disease-causative STING variants could not bind to TBK1 when the STING variants were trapped in the ER. These results demonstrate that the Golgi is an organelle at which STING recruits and activates TBK1 for triggering the STING-dependent type I interferon response. [ABSTRACT FROM AUTHOR]

Stimulator of interferon genes (STING) is an essential molecule for the production of type I interferon (IFN), and other inflammatory cytokines, in response to cytosolic DNA. STING contributes to host defense against infection and anti-tumor responses. Previous reports have demonstrated that STING signaling is required by the adaptor Toll-IL-1 receptor-containing adaptor molecule-1 (TICAM-1), which has been identified as a TLR3-adaptor molecule using mouse embryonic fibroblasts. Here, we demonstrate that TICAM-1 does not affect STING-mediated innate immune responses, as increases in the mRNA expression levels of IFN-β, IL-6, and CCL5 were observed in bone marrow-derived or splenic myeloid cells. Moreover, STING ligand-enhanced co-stimulatory molecule expression, including CD80, CD86, and CD40, was detected on splenic CD11c + DCs, even in Ticam-1 -deficient mice. Our results suggest that STING-mediated innate immune responses and dendritic cell maturation do not require TICAM-1 in myeloid lineage immune cells. TICAM-1 is ubiquitously expressed, even in cell types which do not express TLR3. Therefore, TICAM-1 may possess different functions depending on cell type and signaling purposes. [ABSTRACT FROM AUTHOR]

Cytosolic recognition of DNA has emerged as a critical cellular mechanism of host immune activation upon pathogen invasion. The central cytosolic DNA sensor cGAS activates STING, which is phosphorylated, dimerizes and translocates from the endoplasmic reticulum (ER) to a perinuclear region to mediate IRF-3 activation. Poxviruses are double-stranded DNA viruses replicating in the cytosol and hence likely to trigger cytosolic DNA sensing. Here, we investigated the activation of innate immune signaling by 4 different strains of the prototypic poxvirus vaccinia virus (VACV) in a cell line proficient in DNA sensing. Infection with the attenuated VACV strain MVA activated IRF-3 via cGAS and STING, and accordingly STING dimerized and was phosphorylated during MVA infection. Conversely, VACV strains Copenhagen and Western Reserve inhibited STING dimerization and phosphorylation during infection and in response to transfected DNA and cyclic GMP-AMP, thus efficiently suppressing DNA sensing and IRF-3 activation. A VACV deletion mutant lacking protein C16, thought to be the only viral DNA sensing inhibitor acting upstream of STING, retained the ability to block STING activation. Similar inhibition of DNAinduced STING activation was also observed for cowpox and ectromelia viruses. Our data demonstrate that virulent poxviruses possess mechanisms for targeting DNA sensing at the level of the cGAS-STING axis and that these mechanisms do not operate in replication-defective strains such as MVA. These findings shed light on the role of cellular DNA sensing in poxvirus-host interactions and will open new avenues to determine its impact on VACV immunogenicity and virulence. [ABSTRACT FROM AUTHOR]

Infection of microbial pathogen triggers the innate immune system, and the induction of interferons (IFNs) play a vital role in host antiviral response. Stimulator of interferon genes (STING) was identified as a crucial regulator of the DNA sensing pathway, and activates both nuclear factor-κB and interferon regulatory factor 3 transcription pathways to evoke IFNs production. In this study, we studied the upregulation of STING mRNA expression, induced by IFN-γ in human keratinocytes (HaCaT). STING promoter assays clarified that a gamma-activated sequence (GAS), located at − 7 to − 15-bp, is required for IFN-γ-upregulated promoter activity. Furthermore, an electrophoretic mobility shift assay showed that signal transducers and activators of transcription 1 (STAT1) attach to the GAS motif on the human STING promoter region. This indicates that IFN-γ/Janus kinases/STAT1 signaling is essential for the STING upregulation in human keratinocytes. [ABSTRACT FROM AUTHOR]

Most FDA-approved adjuvants for infectious agents boost humoral but not cellular immunity, and have poorly-understood mechanisms. Stimulator of interferon genes (STING, also known as MITA, MPYS, or ERIS) is an exciting adjuvant target due to its role in cyclic dinucleotide (CDN)-driven anti-viral immunity; however, a major hindrance is STING's cytosolic localization which requires intracellular delivery of its agonists. As a result, STING agonists administered in a soluble form have elicited suboptimal immune responses. Delivery of STING agonists via particle platforms has proven a more successful strategy, but the opportunity for improved formulations and bioactivity remains. In this study we evaluated the adjuvant activity of the potent STING agonist, CDN 3′3′-cGAMP (cGAMP), encapsulated in acid-sensitive acetalated dextran (Ace-DEX) polymeric microparticles (MPs) which passively target antigen-presenting cells for intracellular release. This formulation was superior to all particle delivery systems evaluated and maintained its bioactivity following a sterilizing dose of gamma irradiation. Compared to soluble cGAMP, the Ace-DEX cGAMP MPs enhanced type-I interferon responses nearly 1000-fold in vitro and 50-fold in vivo , caused up to a 10 4 -fold boost in antibody titers, increased Th1-associated responses, and expanded germinal center B cells and memory T cells. Furthermore, the encapsulated cGAMP elicited no observable toxicity in animals and achieved protective immunity against a lethal influenza challenge seven months post-immunization when using CDN adjuvant doses up to 100-fold lower than previous reports. For these reasons, Ace-DEX MP-encapsulated cGAMP represents a potent vaccine adjuvant of humoral and cellular immunity. [ABSTRACT FROM AUTHOR]

Stimulator of interferon gene (STING) plays an important role in the cyclic GMP-AMP synthase (cGAS)-mediated activation of type I IFN responses. In this study, we identified and cloned canine STING gene. Full-length STING encodes a 375 amino acid product that shares the highest similarity with feline STING. Highest levels of mRNA of canine STING were detected in the spleen and lungs while the lowest levels in the heart and muscle. Analysis of its cellular localization showed that STING is localizes to the endoplasmic reticulum. STING overexpression induced the IFN response via the IRF3 and NF-κB pathways and up-regulated the expression of ISG15 and viperin. However, knockdown of STING did not inhibit the IFN-β response triggered by poly(dA:dT), poly(I:C), or SeV. Finally, overexpression of STING significantly inhibited the replication of canine influenza virus H3N2. Collectively, our findings indicate that STING is involved in the regulation of the IFN-β pathway in canine. [ABSTRACT FROM AUTHOR]

Stimulator of interferon genes (STING) is a central and multifaceted mediator in the innate immune response of higher vertebrates. To explore its role in teleost fish, the STING homolog of black carp ( Mylopharyngodon piceus ) (bcSTING) has been cloned and characterized in this paper. bcSTING transcription in Mylopharyngodon piceus fin (MPF) cells increased remarkably in response to GCRV and SVCV infection, or poly (I:C) stimulation. bcSTING migrated around 42 KDa in immunoblot assay and was identified as a cytosolic protein locating on ER majorly through immunofluorescence staining. Under condition of SVCV/GCRV infection or poly (I:C) stimulation, the subcellular distribution of bcSTING majorly displayed on mitochondria, which overlapped with that of bcMAVS. HA-bcSTING instead of bcSTING-HA presented strong IFN-inducing activity in reporter assay and antiviral ability against both SVCV and GCRV in plaque assay. Site mutation of serine (S) on C-terminus of bcSTING demonstrated that both S371 and S379 were crucial for its mediated signaling. Taken together, our study support the conclusion that bcSTING plays an important role in host innate immune defense against RNA virus such as SVCV and GCRV, in which its C-terminus functions crucially. [ABSTRACT FROM AUTHOR]

The STING (Stimulator of Interferon Genes) protein connects microorganism cytosolic sensing with effector functions of the host cell by sensing directly cyclic dinucleotides (CDNs), originating from pathogens or from the host upon DNA recognition. Although STING activation favors effective immune responses against viral infections, its role during bacterial diseases is controversial, ranging from protective to detrimental effects for the host. In this review, we summarize important features of the STING activation pathway and recent highlights about the role of STING in bacterial infections by Chlamydia, Listeria, Francisella , Brucella, Shigella, Salmonella , Streptococcus , and Neisseria genera, with a special focus on mycobacteria. [ABSTRACT FROM AUTHOR]

The equipoise between the disease states of cancer and autoinflammation has perhaps been underappreciated in clinical practice and biomedical research. However, since the discover of STING (stimulator of interferon genes) as an integral regulator of innate immunity, a wealth of information has implicated this signaling pathway in both of these diseases. Under cellular homeostasis, STING serves to detect – and promote immune defense against – DNA viruses and intracellular bacteria, as described in its initial discovery. The role of STING has since been expanded to include tumor surveillance and immune responses to cancer; indeed, defective STING responses are associated with certain cancers. Conversely, constitutive activation of this pathway can result in autoinflammatory disease, whereby STING is over-stimulated by self-DNA. This review explores the current state of STING research, concluding that further elucidation of the details of the STING pathway may offer novel therapeutics for these diseases, which are of considerable clinical gravity. [ABSTRACT FROM PUBLISHER]

Stimulator of interferon genes (STING) is a critical signaling molecule in the innate immune response against DNA viruses by either directly sensing intracellular DNA or functioning as an adaptor molecule to activate the type I interferon (IFN) signaling pathway. We determined the functional interaction between STING and human cytomegalovirus (HCMV). A cDNA library containing 133 HCMV ORFs was screened to identify viral genes that inhibit STING-induced IFN-b promoter activation. Among the screened ORFs, UL122, which encodes the immediate-early 2 86 kDa (IE86) protein, strongly abolished STING-induced IFN-β promoter activation. Interestingly, IE86 protein facilitated the proteasome-dependent degradation of STING and inhibited 2′3′-cGAMP-mediated induction of IFNB1 and CXCL10. Taken together, this study demonstrates the existence of a post-translational regulation of STING by HCMV IE86 protein. [ABSTRACT FROM AUTHOR]

The stimulator of interferon (IFN) genes (STING) is a broad antimicrobial factor that restricts herpes simplex virus (HSV) by activating type I interferon and proinflammatory responses upon sensing of foreign DNA. UL46 is one of the most abundant tegument proteins of HSV-1, but a well-established function has yet to be found. We found that the HSV-1 UL46 protein interacts with and colocalizes with STING. A ▵ UL46 virus displayed growth defects and activated innate immunity, but both effects were alleviated in STING knockdown cells. UL46 was also required for the inhibition of the 2',3'-cyclic GMP-AMP (cGAMP)-dependent immune responses during infection. In cells expressing UL46, out of the context of the infection, innate immunity to a ▵ ICP0 virus was largely compromised, and that permitted ICP0-deficient mutants to replicate. The UL46-expressing cell lines also rescued the defects of the ▵ UL46 virus and enhanced wild-type virus infection. The UL46-expressing cell lines did not activate interferon-stimulated gene (ISG) transcription following treatment with the noncanonical cyclic dinucleotide 2',3'-cGAMP, suggesting that the STING pathway may be compromised. Indeed, we found that both proteins STING and IFI16 were eliminated in cells constitutively expressing UL46 and that the accumulation of their transcripts was blocked. Finally, we demonstrated that UL46 via its N terminus binds to STING and, via its C terminus, to TBK1. These interactions appear to modulate the functions of STING during HSV-1 infection. Taken together, our studies describe a novel function for one of the least-studied proteins of HSV, the tegument protein UL46 and that function involves the evasion of foreign DNA-sensing pathways. IMPORTANCE Herpes simplex virus 1 (HSV-1) afflicts 80% of the population worldwide, causing various diseases. After initial infection, the virus establishes latent reservoirs in sensory neurons and persists for life. Here we describe novel interactions between HSV-1 and the DNA sensor STING. We found that (i) HSV-1 tegument protein UL46 interacts with and colocalizes with STING; (ii) UL46 expressed out of the context of the infection blocks type I interferon triggered by STING stimuli, through the elimination of STING and of interferon-inducible protein 16 (IFI16); (iii) a ▵ UL46 virus displayed growth defects, which were rescued in STING knockdown cells; (iv) the ▵ UL46 virus failed to block innate immunity triggered by ligands of STING such as 2',3'-cGAMP and also activated IFN-β and ISG expression; and (v) UL46 binds to both STING and TBK1 through different domains. We conclude that UL46 counteracts the actions of STING during HSV-1 infection. [ABSTRACT FROM AUTHOR]

Stimulator of interferon gene ( sting) was identified and characterized from common carp Cyprinus carpio. The sting messenger (m) RNA encoded a polypeptide of 402 amino acids with a calculated molecular mass of 46·184 kDa and an isoelectronic point of 6·08. The deduced protein of sting contained a signal peptide, three transmembrane motifs in the N-terminal region and four putative motifs ( RXR) found in resident endoplasmic reticulum proteins. mRNA expression of sting was present in twelve investigated tissues, and was up-regulated by koi herpesvirus ( KHV) in vivo and in vitro. The transcription of sting was altered by poly(I:C) and poly( dT: dA) stimulation in vitro. The findings suggested that sting is an inducible gene involved in innate immunity against DNA- and RNA-derived pathogens. To investigate defence mechanisms in C. carpio development, sting level in embryos, larvae and juvenile fish was monitored following KHV challenge. The sting message was negligible in embryos prior to hatching, but observed at higher transcriptional levels throughout larval and juvenile stages. Investigation showed the mRNA expression profiles of genes encoding for proteins promoting various functions in the interferon pathway, from pattern recognition receptors to antiviral genes, to be significantly induced in all examined organs by in vivo infection with KHV. Following KHV infection, the ifn message was significantly downregulated in spleen, head kidney, brain and hepatopancreas but notably up-regulated in gill, intestine and skin, suggesting that ifn induction might be related to the mucosal immune system and virus anti- ifn mechanisms. These results provided the basis for further research into the role and mechanisms of sting in fishes. [ABSTRACT FROM AUTHOR]

Therapeutic vaccines to induce anti-tumor CD8 T cells have been used in clinical trials for advanced melanoma patients, but the clinical response rate and overall survival time have not improved much. We believe that these dismal outcomes are caused by inadequate number of antigen-specific CD8 T cells generated by most vaccines. In contrast, huge CD8 T cell responses readily occur during acute viral infections. High levels of type-I interferon (IFN-I) are produced during these infections, and this cytokine not only exhibits anti-viral activity but also promotes CD8 T cell responses. The studies described here were performed to determine whether promoting the production of IFN-I could enhance the potency of a peptide vaccine. We report that cyclic diguanylate monophosphate (c-di-GMP), which activates the stimulator of interferon genes, potentiated the immunogenicity and anti-tumor effects of a peptide vaccine against mouse B16 melanoma. The synergistic effects of c-di-GMP required co-administration of costimulatory anti-CD40 antibody, the adjuvant poly-IC, and were mediated in part by IFN-I. These findings demonstrate that peptides representing CD8 T cell epitopes can be effective inducers of large CD8 T cell responses in vaccination strategies that mimic acute viral infections. [ABSTRACT FROM AUTHOR]

Background: Traumatic brain injury (TBI) represents a major cause of disability and death worldwide with sustained neuroinflammation and autophagy dysfunction contributing to the cellular damage. Stimulator of interferon genes (STING)-induced type-I interferon (IFN) signalling is known to be essential in mounting the innate immune response against infections and cell injury in the periphery, but its role in the CNS remains unclear. We previously identified the type-I IFN pathway as a key mediator of neuroinflammation and neuronal cell death in TBI. However, the modulation of the type-I IFN and neuroinflammatory responses by STING and its contribution to autophagy and neuronal cell death after TBI has not been explored.Methods: C57BL/6J wild-type (WT) and STING-/- mice (8-10-week-old males) were subjected to controlled cortical impact (CCI) surgery and brains analysed by QPCR, Western blot and immunohistochemical analyses at 2 h or 24 h. STING expression was also analysed by QPCR in post-mortem human brain samples.Results: A significant upregulation in STING expression was identified in late trauma human brain samples that was confirmed in wild-type mice at 2 h and 24 h after CCI. This correlated with an elevated pro-inflammatory cytokine profile with increased TNF-α, IL-6, IL-1β and type-I IFN (IFN-α and IFN-β) levels. This expression was suppressed in the STING-/- mice with a smaller lesion volume in the knockout animals at 24 h post CCI. Wild-type mice also displayed increased levels of autophagy markers, LC3-II, p62 and LAMP2 after TBI; however, STING-/- mice showed reduced LAMP2 expression suggesting a role for STING in driving dysfunctional autophagy after TBI.Conclusion: Our data implicates a detrimental role for STING in mediating the TBI-induced neuroinflammatory response and autophagy dysfunction, potentially identifying a new therapeutic target for reducing cellular damage in TBI. [ABSTRACT FROM AUTHOR]

Summary The ability of dying cells to activate antigen-presenting cells (APCs) is carefully controlled to avoid unwarranted inflammatory responses. Here, we show that engulfed cells containing cytosolic double-stranded DNA species (viral or synthetic) or cyclic di-nucleotides (CDNs) are able to stimulate APCs via extrinsic STING (stimulator of interferon genes) signaling, to promote antigen cross-presentation. In the absence of STING agonists, dying cells were ineffectual in the stimulation of APCs in trans . Cytosolic STING activators, including CDNs, constitute cellular danger-associated molecular patterns (DAMPs) only generated by viral infection or following DNA damage events that rendered tumor cells highly immunogenic. Our data shed insight into the molecular mechanisms that drive appropriate anti-tumor adaptive immune responses, while averting harmful autoinflammatory disease, and provide a therapeutic strategy for cancer treatment. [ABSTRACT FROM AUTHOR]