Ectopic expression of cGAS in Salmonella typhimurium enhances STING-mediated IFN-β response in human macrophages and dendritic cells.

Interferon (IFN)-β induction via activation of the stimulator of interferon genes (STING) pathway has shown promising results in tumor models. STING is activated by cyclic dinucleotides such as cyclic GMP-AMP dinucleotides with phosphodiester linkages 2'-5' and 3'-5' (cGAMPs), that are produced by cyclic GMP-AMP synthetase (cGAS). However, delivery of STING pathway agonists to the tumor site is a challenge. Bacterial vaccine strains have the ability to specifically colonize hypoxic tumor tissues and could therefore be modified to overcome this challenge. Combining high STING-mediated IFN-β levels with the immunostimulatory properties of Salmonella typhimurium could have potential to overcome the immune suppressive tumor microenvironment. We have engineered S. typhimurium to produce cGAMP by expression of cGAS. The ability of cGAMP to induce IFN-β and its IFN-stimulating genes was addressed in infection assays of THP-I macrophages and human primary dendritic cells (DCs). Expression of catalytically inactive cGAS is used as a control. DC maturation and cytotoxic T-cell cytokine and cytotoxicity assays were conducted to assess the potential antitumor response in vitro. Finally, by making use of different S. typhimurium type III secretion (T3S) mutants, the mode of cGAMP transport was elucidated. Expression of cGAS in S. typhimurium results in a 87-fold stronger IFN-β response in THP-I macrophages. This effect was mediated by cGAMP production and is STING dependent. Interestingly, the needle-like structure of the T3S system was necessary for IFN-β induction in epithelial cells. DC activation included upregulation of maturation markers and induction of type I IFN response. Coculture of challenged DCs with cytotoxic T cells revealed an improved cGAMP-mediated IFN-γ response. In addition, coculture of cytotoxic T cells with challenged DCs led to improved immune-mediated tumor B-cell killing. S. typhimurium can be engineered to produce cGAMPs that activate the STING pathway in vitro. Furthermore, they enhanced the cytotoxic T-cell response by improving IFN-γ release and tumor cell killing. Thus, the immune response triggered by S. typhimurium can be enhanced by ectopic cGAS expression. These data show the potential of S. typhimurium-cGAS in vitro and provides rationale for further research in vivo.

Waanders L ，van der Donk LEH ，Ates LS ，Maaskant J ，van Hamme JL ，Eldering E ，van Bruggen JAC ，Rietveld JM ，Bitter W ，Geijtenbeek TBH ，Kuijl CP ... -  《Journal for ImmunoTherapy of Cancer》

Salmonella Induces the cGAS-STING-Dependent Type I Interferon Response in Murine Macrophages by Triggering mtDNA Release.

Xu L ，Li M ，Yang Y ，Zhang C ，Xie Z ，Tang J ，Shi Z ，Chen S ，Li G ，Gu Y ，Wang X ，Zhang F ，Wang Y ，Shen X ... -  《mBio》

Triggering of the cGAS-STING Pathway in Human Plasmacytoid Dendritic Cells Inhibits TLR9-Mediated IFN Production.

Plasmacytoid dendritic cells (pDCs) are potent producers of type I and type III IFNs and play a major role in antiviral immunity and autoimmune disorders. The innate sensing of nucleic acids remains the major initiating factor for IFN production by pDCs. TLR-mediated sensing of nucleic acids via endosomal pathways has been studied and documented in detail, whereas the sensing of DNA in cytosolic compartment in human pDCs remains relatively unexplored. We now demonstrate the existence and functionality of the components of cytosolic DNA-sensing pathway comprising cyclic GMP-AMP (cGAMP) synthase (cGAS) and stimulator of IFN gene (STING) in human pDCs. cGAS was initially located in the cytosolic compartment of pDCs and time-dependently colocalized with non-CpG double-stranded immunostimulatory DNA (ISD). Following the colocalization of ISD with cGAS, the downstream pathway was triggered as STING disassociated from its location at the endoplasmic reticulum. Upon direct stimulation of pDCs by STING agonist 2'3' cGAMP or dsDNA, pDC-s produced type I, and type III IFN. Moreover, we documented that cGAS-STING-mediated IFN production is mediated by nuclear translocation of IRF3 whereas TLR9-mediated activation occurs through IRF7. Our data also indicate that pDC prestimulation of cGAS-STING dampened the TLR9-mediated IFN production. Furthermore, triggering of cGAS-STING induced expression of SOCS1 and SOCS3 in pDCs, indicating a possible autoinhibitory loop that impedes IFN production by pDCs. Thus, our study indicates that the cGAS-STING pathway exists in parallel to the TLR9-mediated DNA recognition in human pDCs with cross-talk between these two pathways.

Deb P ，Dai J ，Singh S ，Kalyoussef E ，Fitzgerald-Bocarsly P ... -  《-》

Cancer-Cell-Intrinsic cGAS Expression Mediates Tumor Immunogenicity.

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.

Schadt L ，Sparano C ，Schweiger NA ，Silina K ，Cecconi V ，Lucchiari G ，Yagita H ，Guggisberg E ，Saba S ，Nascakova Z ，Barchet W ，van den Broek M ... -  《Cell Reports》

Human plasmacytoid dentritic cells elicit a Type I Interferon response by sensing DNA via the cGAS-STING signaling pathway.

Plasmacytoid dendritic cells (pDCs) are a major source of type I interferon (IFN) and are important for host defense by sensing microbial DNA via TLR9. pDCs also play a critical role in the pathogenesis of IFN-driven autoimmune diseases. Yet, this autoimmune reaction is caused by the recognition of self-DNA and has been linked to TLR9-independent pathways. Increasing evidence suggests that the cytosolic DNA receptor cyclic GMP-AMP (cGAMP) synthase (cGAS) is a critical component in the detection of pathogens and contributes to autoimmune diseases. It has been shown that binding of DNA to cGAS results in the synthesis of cGAMP and the subsequent activation of the stimulator of interferon genes (STING) adaptor to induce IFNs. Our results show that the cGAS-STING pathway is expressed and activated in human pDCs by cytosolic DNA leading to a robust type I IFN response. Direct activation of STING by cyclic dinucleotides including cGAMP also activated pDCs and knockdown of STING abolished this IFN response. These results suggest that pDCs sense cytosolic DNA and cyclic dinucleotides via the cGAS-STING pathway and that targeting this pathway could be of therapeutic interest.

Bode C ，Fox M ，Tewary P ，Steinhagen A ，Ellerkmann RK ，Klinman D ，Baumgarten G ，Hornung V ，Steinhagen F ... -  《-》