Littérature scientifique sur le sujet « STING Knockout »

ObjectiveDespite the importance of type I interferon (IFN-I) in systemic lupus erythematosus (SLE) pathogenesis, the mechanisms of IFN-I production have not been fully elucidated. Recognition of nucleic acids by DNA sensors induces IFN-I and interferon-stimulated genes (ISGs), but the involvement of cyclic guanosine monophosphate (GMP)–AMP synthase (cGAS) and stimulator of interferon genes (STING) in SLE remains unclear. We studied the role of the cGAS–STING pathway in the IFN-I-producing cascade driven by SLE serum.MethodsWe collected sera from patients with SLE (n=64), patients with other autoimmune diseases (n=31) and healthy controls (n=35), and assayed them using a cell-based reporter system that enables highly sensitive detection of IFN-I and ISG-inducing activity. We used Toll-like receptor-specific reporter cells and reporter cells harbouring knockouts of cGAS, STING and IFNAR2 to evaluate signalling pathway-dependent ISG induction.ResultsIFN-I bioactivity and ISG-inducing activities of serum were higher in patients with SLE than in patients with other autoimmune diseases or healthy controls. ISG-inducing activity of SLE sera was significantly reduced in STING-knockout reporter cells, and STING-dependent ISG-inducing activity correlated with disease activity. Double-stranded DNA levels were elevated in SLE. Apoptosis-derived membrane vesicles (AdMVs) from SLE sera had high ISG-inducing activity, which was diminished in cGAS-knockout or STING-knockout reporter cells.ConclusionsAdMVs in SLE serum induce IFN-I production through activation of the cGAS–STING pathway. Thus, blockade of the cGAS–STING axis represents a promising therapeutic target for SLE. Moreover, our cell-based reporter system may be useful for stratifying patients with SLE with high ISG-inducing activity.

Stimulator of interferon genes (STING) is an adapter protein that is activated when cyclic dinucleotides (CDNs) are present. CDNs originate from the cytosolic DNA of both pathogens and hosts. STING activation promotes efficient immune responses against viral infections; however, its impact in bacterial infections is unclear. In this study, we investigated the role of Sting in bacterial infections by successfully creating a sting-deficient (sting(−/−) with a 4-bp deletion) knockout zebrafish model using CRISPR/Cas9. The transcriptional modulation of genes downstream of cGAS (cyclic GMP-AMP synthase)-Sting pathway-related genes was analyzed in seven-day-old wild-type (WT) and sting(−/−) embryos, as well as in four-day-old LPS-stimulated embryos. The expression of downstream genes was higher in sting(−/−) than in healthy WT fish. The late response was observed in sting(−/−) larvae following LPS treatment, demonstrating the importance of Sting-induced immunity during bacterial infection by activating the cGAS–STING pathway. Furthermore, adult sting(−/−) fish had a high mortality rate and significantly downregulated cGAS–STING pathway-related genes during Edwardsiella piscicida (E. piscicida) infection. In addition, we assessed NF-κB pathway genes following E. piscicida infection. Our results show fluctuating patterns of interleukin-6 (il6) and tumor necrosis factor-α (tnfα) expression, which is likely due to the influence of other NF-κB pathway-related immune genes. In summary, this study demonstrates the important role of Sting against bacterial infection.

The dichotomic contribution of cancer cell lysis and tumor immunogenicity is considered essential for effective oncovirotherapy, suggesting that the innate antiviral immune response is a hurdle for efficacy of oncolytic viruses. However, emerging evidence is resizing this view. By sensing cytosolic DNA, the cyclic GMP-AMP synthase (cGAS) and stimulator of interferon genes (STING) axis can both counteract viral spread and contribute to the elicitation of adaptive immunity via type I interferon responses. In this paper, we analyzed the tumor-resident function of Sting-mediated DNA sensing in a combined approach of oncovirotherapy and PD-1 immune checkpoint blockade, in an immunocompetent murine model. While supporting increased lytic potential by oncolytic HER2-retargeted HSV-1 in vitro and in vivo, Sting-knockout tumors showed molecular signatures of an immunosuppressive tumor microenvironment. These signatures were correspondingly associated with ineffectiveness of the combination therapy in a model of established tumors. Results suggest that the impairment in antiviral response of Sting-knockout tumors, while favoring viral replication, is not able to elicit an adequate immunotherapeutic effect, due to lack of immunogenic cell death and the inability of Sting-knockout cancer cells to promote anti-tumor adaptive immune responses. Accordingly, we propose that antiviral, tumor-resident Sting provides fundamental contributions to immunotherapeutic efficacy of oncolytic viruses.

The innate immune system is the first line of host defense sensing viral infection. Manganese (Mn) has recently been found to be involved in the activation of the innate immune DNA-sensing cGAS-STING pathway and subsequent anti-DNA virus function. However, it is still unclear whether Mn2+ mediates host defense against RNA viruses. In this study, we demonstrate that Mn2+ exhibited antiviral effects against various animal and human viruses, including RNA viruses such as PRRSVs and VSV, as well as DNA viruses such as HSV1, in a dose-dependent manner. Moreover, cGAS and STING were both investigated in the Mn2+ mediated antiviral roles using the knockout cells made by the CRISPR-Cas9 approach. Unexpectedly, the results revealed that neither cGAS knockout nor STING knockout had any effect on Mn2+-mediated antiviral functions. Nevertheless, we verified that Mn2+ promoted the activation of the cGAS-STING signaling pathway. These findings suggest that Mn2+ has broad-spectrum antiviral activities in a cGAS-STING pathway independent manner. This study also provides significant insights into redundant mechanisms participating in the Mn2+ antiviral functions, and also indicates a new target for Mn2+ antiviral therapeutics.

ABSTRACTInfection by adenovirus, a nonenveloped DNA virus, induces antiviral innate and adaptive immune responses. Studies of transformed human and murine cell lines using short hairpin RNA (shRNA) knockdown strategies identified cyclic guanine adenine synthase (cGAS) as a pattern recognition receptor (PRR) that contributes to the antiadenovirus response. Here we demonstrate how the cGAS/STING cascade influences the antiviral innate and adaptive immune responses in a murine knockout model. Using knockout bone marrow-derived dendritic cells (BMDCs) and bone marrow-derived macrophages (BMMOs), we determined that cGAS and STING are essential to the induction of the antiadenovirus response in these antigen-presenting cells (APCs)in vitro. We next determined how the cGAS/STING cascade impacts the antiviral response following systemic administration of a recombinant adenovirus type 5 vector (rAd5V). Infection of cGAS−/−and STING−/−mice results in a compromised early antiviral innate response compared to that in wild-type (WT) controls: significantly lower levels of beta interferon (IFN-β) secretion, low levels of proinflammatory chemokine induction, and reduced levels of antiviral transcript induction in hepatic tissue. At 24 h postinfection, levels of viral DNA and reporter gene expression in the liver were similar in all strains. At 28 days postinfection, clearance of infected hepatocytes in cGAS or STING knockout mice was comparable to that in WT C57BL/6 mice. Levels of neutralizing anti-Ad5V antibody were modestly reduced in infected cGAS mice. These data support a dominant role for the cGAS/STING cascade in the early innate antiviral inflammatory response to adenovirus vectors. However, loss of the cGAS/STING pathway did not affect viral clearance, and cGAS deficiency had a modest influence on the magnitude of the antiviral humoral immune response to adenovirus infections.IMPORTANCEThe detection of viral infection by host sentinel immune cells contributes to the activation of a complex and varied antiviral innate and adaptive immune response, which limits virus replication, spread, and susceptibility to infection. In this study, we have characterized how the cGAS/STING DNA-sensing cascade contributes to early detection of adenovirus infections. cGAS influences APC activation and early innate antiviral inflammatory immune responses, but adaptive immune pathways associated with virus clearance and anti-Ad antibody production were minimally influenced by the loss of the cGAS PRR signaling cascade.

Background: Malignant pleural mesothelioma (MESO) has a poor prognosis despite aggressive treatment with surgery, radiation and chemotherapy, and novel therapeutic approaches are needed. IRF3 is a downstream molecule of the cGAS/STING signaling pathway, but its roles have not been investigated in MESO. Methods: Various murine mesothelioma cell lines were inoculated into wild type (WT) and IRF3 knockout (IRF3KO) mice to compare tumor growth. AE17-bearing mice were treated with local radiotherapy (LRT) to evaluate the effect on tumor growth, and immune cell infiltration was analyzed by flow cytometry 20 days after tumor inoculation. TCGA data were used to examine the relationship between mRNA expression of IRF3 and genes of the cGAS/STING signaling cascade on prognosis in MESO. Correlations between gene expression of IRF3, cGAS/STING signaling pathway, and immune checkpoints were analyzed in TCGA MESO and our scRNA-Seq data from MESO patients. Results: In mouse mesothelioma models, AK7, RN5 and ZiP3 were completely rejected in IRF3KO mice 20 days after the tumor challenge. AE17tumor volume was slightly larger than WT mice around day 10 before shrinking and becoming significantly smaller than WT mice on day 20. LRT accelerated tumor shrinkage of AE17 tumors in IRF3KO mice. Compared with WT mice, the number of macrophages infiltrating the tumor of IRF3KO mice was significantly reduced, and CD4+ T cells and CD8+IFNγ+ T cells were significantly increased. TCGA data showed that IRF3 expression was an unfavorable prognostic factor in MESO patients. IRF3 expression, the cGAS/STING signaling pathway, and immune checkpoints were positively correlated. Conclusion: IRF3 could play a critical role in the tumor immune microenvironment of MESO.

Porcine reproductive and respiratory syndrome virus (PRRSV) modulates host innate immunity which plays a key role against PRRSV infection. As a RNA virus, PRRSV is mainly sensed by innate immune RNA receptors, whereas the role of innate immune DNA sensors in the PRRSV infection has not been elucidated. Here, we investigated the roles of DNA sensing cGAS-STING pathway in both PRRSV infected Marc-145 cells and porcine macrophages. The results show that in Marc-145 cells, the stable expression of STING with or without stimulations exhibited anti-PRRSV activity, and STING knockout heightened PRRSV infection. In CD163-3D4/21 porcine macrophages, either expression of STING or stimulation of cGAS-STING signaling obviously suppressed PRRSV infection, whereas in STING knockdown macrophages, the PRRSV infection was upregulated. Our results clearly demonstrate that the host cGAS-STING signal exerts an important antiviral role in PRRSV infection.

It is well known that plasmid DNA transfection, prior to virus infection, negatively affects infection efficiency. Here, we show that cytosolic plasmid DNA activates the cGAS/STING signaling pathway, which ultimately leads to the induction of an antiviral state of the cells. Using a transient one-plasmid clustered regularly interspaced short palindromic repeat (CRISPR)/Cas9 system, we generated cGAS/STING-knockout cells and show that these cells can be infected after plasmid DNA transfection as efficiently as nontransfected cells.

Abstract Background: The stimulator of interferon genes (STING) plays a central role in innate immune response against infection and cancer. Several cyclic di-nucleotide (CDN) and non-CDN small molecule STING agonists have demonstrated effectiveness against cancer in preclinical animal models, however their clinical trials showed limited therapeutic efficacy. ONM-501, a dual-activating STING agonist employs PC7A, a synthetic polymer that induces polyvalent STING condensation and prolongs innate immune activation has been recently developed. ONM-501 encapsulates the endogenous STING agonist cGAMP with the PC7A micelles offering dual ‘burst’ and ‘sustained’ STING activation. The mechanism and effectiveness of intratumorally delivered ONM-501 as an immunotherapy against solid tumors has been demonstrated in preclinical models. Methods: ONM-501 was recently evaluated for STING activation across different species: STING related IFNB1 and CXCL10 gene expression after ONM-501 treatment was measured by RT-qPCR in PBMCs from rat, cynomolgus monkey, and human. ONM-501 antitumor efficacy was evaluated in murine syngeneic tumor models. Abscopal effect was demonstrated by studying antitumor efficacy using both a primary/distal model and a lung metastatic model. STING knockout mice and STING knockout cancer cells were used to clarify the dependence of STING status to the ONM-501 antitumor immunity. Immune cell dependence was further elucidated by depletion of specific immune cell populations. Pilot safety studies including major organ function and systemic cytokine levels were performed in immunocompetent mice. Results: STING activation was observed across different species by measuring IFNB1 and CXCL10 mRNA in PBMCs from rat, cynomolgus monkey, and human after ONM-501 treatment. Antitumor efficacy was demonstrated both as a monotherapy and in combination with anti-PD1 in six different syngeneic tumor models. ONM-501 also induced an abscopal effect - tumor inhibition was observed in both primary and distal MC38 tumors in the same animal. Reduction of lung metastasis in an immune “cold” triple negative orthotopic breast cancer 4T1 model further confirmed the systemic antitumor immunity. Knocking out host STING (i.e. STING KO mice) rather than cancer cell STING KO resulted in abrogation of tumor inhibition suggesting that the host STING status is responsible for ONM-501-mediated anti-tumor immunity. Immune cell depletion studies further clarified that the antitumor immunity is dependent on CD8+ T cell and dendritic cells (DCs). No alteration of main organ function or systemic cytokine storm were observed in the pilot safety study. Conclusions: ONM-501 demonstrated marked anti-tumor efficacy in a panel of syngeneic tumor models. The anti-tumor effect was mediated by host STING and dependent on CD8+ T cells and DCs. These results support further evaluation of ONM-501 as a clinical candidate for the potential treatment of solid tumors. Citation Format: Suxin Li, Jian Wang, Jonathan Wilhelm, Qingtai Su, Gaurav Bharadwaj, Jason Miller, Wei Li, Katy Torres, Ruolan Han, Tian Zhao, Jinming Gao. ONM-501: A polyvalent STING agonist for oncology immunotherapy [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2022; 2022 Apr 8-13. Philadelphia (PA): AACR; Cancer Res 2022;82(12_Suppl):Abstract nr 4234.

Oncolytic viruses promote anti-tumour immune response by direct tumour cell killing and activation of intratumoural immune system. The role of innate antiviral immune response to oncolytic viruses is still debated, as they counteract viral replication and trigger adaptive antitumor immunity. The DNA sensing-mediated cGAS/STING axis may act as a key balancer between lytic and immunotherapeutic activity of oncolytic viruses. Indeed, upon infection, viral DNA is sensed by cGAS/STING axis that, in turn, induces type-I interferon cascade counteracting viral replication and spread. For this reason, STING represents a hurdle for classical lytic-centric function of oncolytic viruses. On the other side, the immunological role of STING should also be considered, as it is emerging as a key bridge between innate and adaptive immunity. To evaluate the role of STING expression in tumour cells in response to onco-virotherapy, we generated murine STING KO tumour cell lines through CRISPR/Cas9 genome editing. Preclinical studies in syngeneic immunocompetent tumour-bearing mice showed that the inactivation of STING in tumour cells, while favouring oncolytic viral replication, impaired the immunotherapeutic effects of combination therapy based on herpetic oncolytic virus and PD1 blockade. Molecular characterization of tumours revealed that loss of STING prevents antitumour immune activation inducing a tolerogenic cell death and immunosuppressive tumour microenvironment. Accordingly, I propose that antiviral, tumourresident STING provides fundamental contributions to heat-up the TME eliciting immunotherapeutic efficacy of oncolytic viruses.

Hi-Spot are highly re-aggregated neural cultures grown on PTFE membrane at the liquidair interface. The Hi-Spot protocol was developed by scientists at Capsant Neurotechnologies using embryonic neural tissue. We characterised Hi-Spots made using existing protocols to confirm they represented a maturing neural network with molecular, cellular and functional signatures. We have additionally modified existing protocols to allow use of postnatal tissue as the source for dissociation and re-aggregation. Hi-Spots made from postnatal day zero (P0) rats self organised into a complex 3D tissue-like structure containing anatomically synaptically-interconnected neurons, astroglia and microglia. This CNS analogue of brain tissue provides for the emergence of a co-ordinated excitatory and inhibitory network, demonstrating a maturing pattern of activity involving single spikes developing into bursting behaviour driven by intrinsic synaptic activity. This activity can be represented as frequency or averaged amplitude (RMS), increases with time in culture and is blocked by glutamate receptor antagonists and stimulated by the inhibitory receptor antagonist bicuculline. The validity of its more in vivo-like organization, not observed in more conventional dissociated cultures, is evidenced by a glutamate toxicity resistance in the Hi-Spot cultures. Further, the thesis describes the optimisations to the Hi-Spot protocol to allow viable high density re-aggregated cultures to be made from individual rather than pooled brain dissociations. Modifications to the protocol included dissociation of the mouse tissue using a protease papain kit and cell plating at an increased cell density. Using this as a platform we have gone on to investigate the neuronal dysfunction occurring in Cysteine String Protein (CSP)α-/- mice. CSPα is a presynaptic protein thought to have co-chaperone like functions, mice lacking CSPα are born alive but show progressive weakness and neuronal degeneration soon after birth (Fernandez-Chacon, Wolfel et al. 2004). Hi-Spots formed from CSPα -/- tissue did not show overt neurodegenerative characteristics compared to +/+ controls and functional analysis demonstrated that at~DIV14 CSPα +/+ and -/- mice displayed equal levels of basal spontaneous network activity. Addition of bicuculline (50μM) to +/+ cultures lead to a significantly increasedfrequency and RMS value. However, in -/- cultures there was no increase induced by bicuculline. This may be due to an inability of CSPα -/- cultures to sustain high frequency synaptic transmission that is associated with bursting activity, or a selective degeneration of a sub-population of inhibitory neurons and a homeostatic network plasticity. The data suggest CSPα may act to protect the ability of neurons for high frequency synaptic transmission and/ or protect inhibitory neurons from degeneration.