cyclic-guanosine-monophosphate-adenosine-monophosphate and Disease-Models--Animal

Topics: Amyotrophic Lateral Sclerosis; Animals; Disease Models, Animal; DNA-Binding Proteins; Humans; Inflammation; Membrane Proteins; Mice; Nucleotides, Cyclic; Nucleotidyltransferases

Stimulator of interferon genes (STING) promotes anti-tumour immunity by linking innate and adaptive immunity, but it remains unclear how intratumoural treatment with STING agonists yields anti-tumour effects. Here we demonstrate that intratumoural injection of the STING agonist cGAMP induces strong, rapid, and selective apoptosis of tumour endothelial cells (ECs) in implanted LLC tumour, melanoma and breast tumour, but not in spontaneous breast cancer and melanoma. In both implanted and spontaneous tumours, cGAMP greatly increases TNFα from tumour-associated myeloid cells. However, compared to spontaneous tumour ECs, implanted tumour ECs are more vulnerable to TNFα-TNFR1 signalling-mediated apoptosis, which promotes effective anti-tumour activity. The spontaneous tumour's refractoriness to cGAMP is abolished by co-treatment with AKT 1/2 inhibitor (AKTi). Combined treatment with cGAMP and AKTi induces extensive tumour EC apoptosis, leading to extensive tumour apoptosis and marked growth suppression of the spontaneous tumour. These findings propose an advanced avenue for treating primary tumours that are refractory to single STING agonist therapy.

Topics: Animals; Antineoplastic Combined Chemotherapy Protocols; Apoptosis; Cell Line, Tumor; Disease Models, Animal; Drug Resistance, Neoplasm; Female; Gene Knockdown Techniques; Human Umbilical Vein Endothelial Cells; Humans; Immunity, Innate; Injections, Intralesional; Male; Membrane Proteins; Mice; Mice, Transgenic; Neoplasms; Nucleotides, Cyclic; Phosphorylation; Proto-Oncogene Proteins c-akt; Receptors, Tumor Necrosis Factor, Type I; Signal Transduction; Tumor Microenvironment; Tumor Necrosis Factor-alpha

The process of repair after skin injury is precisely regulated by a variety of mediators such as cytokines and chemokines. Recent reports demonstrated that cytoplasmic DNA-sensor cyclic GMP-AMP synthase (cGAS) activates the stimulator of interferon genes (STING) via production of cyclic GMP-AMP (cGAMP) and subsequently induces inflammatory cytokines, including type I interferon (IFN).. We examined whether activation of the STING pathway by cGAMP affects the process of skin wound repair.. The skin wound repair model was established using wild-type (WT) mice. Two full-thickness skin biopsies were taken from the right and left subscapular regions. One site was treated with ointment containing cGAMP, and the other was treated with a control ointment. Changes in wound size over time were calculated using photography.. Treatment with cGAMP significantly accelerated skin wound healing up to day 6. Biochemical analyses showed that topical treatment with cGAMP on wound sites promoted STING signaling pathway and enhanced the expression of IFN-β, CXCL10 and CCL2 in the wound sites treated with cGAMP markedly compared with the control. The scratch assay also revealed that cGAMP treatment accelerated wound closure in mouse embryonic fibroblasts. The acceleration of skin wound repair by cGAMP in WT mouse was impaired by administration of anti-IFNR antibody and anti-CXCR3 antibody respectively.. These results revealed that topical treatment with cGAMP accelerates skin wound healing by inducing type I IFN and CXCL10/CXCR3. Topical administration of cGAMP might contribute to new effective treatments for accelerating skin wound healing.

Topics: Animals; Disease Models, Animal; Humans; Interferon Type I; Male; Membrane Proteins; Mice; Nucleotides, Cyclic; Receptor, Interferon alpha-beta; Receptors, CXCR3; Signal Transduction; Skin; Wound Healing

Intracellular DNA triggers interferon release during the innate immune response. Cyclic GMP-AMP synthase (cGAS) senses intracellular double-stranded DNA not only in response to viral infection but also under autoimmune conditions. Measuring the levels of cyclic GMP-AMP (cGAMP) as a second messenger of cGAS activation is important to elucidate the physiological and pathological roles of cGAS. Therefore, we generated monoclonal antibodies against cGAMP using hybridoma technology to test antibody specificity and establish methods to detect intracellular cGAMP. The resulting cGAMP-specific antibody enabled the development of a time-resolved fluorescence energy transfer assay with a quantifiable range of 0.1 nM to 100 nM cGAMP. Using this assay, we detected cellular and tissue cGAMP. We confirmed that the cGAMP antibody successfully targeted intracellular cGAMP through immunocytochemical analyses. These results demonstrated that the cGAMP antibody is a powerful tool that allows determining cGAS involvement in autoimmunity and disease pathology at the cell and tissue levels.

Topics: Animals; Antibodies, Monoclonal; Antibody Specificity; Autoimmune Diseases of the Nervous System; Autoimmunity; Biomarkers; Caco-2 Cells; Disease Models, Animal; Enzyme Activation; Exodeoxyribonucleases; Fluorescence Resonance Energy Transfer; HEK293 Cells; High-Throughput Screening Assays; HL-60 Cells; Humans; Immunohistochemistry; Mice, Inbred C57BL; Mice, Knockout; Neoplasms; Nervous System Malformations; Nucleotides, Cyclic; Nucleotidyltransferases; Phosphoproteins; Predictive Value of Tests; Reproducibility of Results

cGAS is a sensor of cytosolic DNA and responds equally to exogenous and endogenous DNA. After recognition of cytosolic dsDNA or ssDNA, cGAS synthesizes the second messenger 2'3'-cGAMP, which then binds to and activates stimulator of interferon genes (STING). STING plays an essential role in responding to pathogenic DNA and self-DNA in the context of autoimmunity. In pathologic conditions, such as stroke or hypoxia-ischemia (HI), DNA can gain access into the cytoplasm of the cell and leak from the dying cells into the extracellular environment, which potentially activates cGAS/STING. Recent in vivo studies of myocardial ischemia, traumatic brain injury, and liver damage models suggest that activation of cGAS/STING is not only a side-effect of the injury, but it can also actively contribute to cell death and apoptosis. We found, for the first time, that cGAS/STING pathway becomes activated between 24 and 48 h after HI in a 10-day-old rat model. Silencing STING with siRNA resulted in decreased infarction area, reduced cortical neurodegeneration, and improved neurobehavior at 48 h, suggesting that STING can contribute to injury progression after HI. STING colocalized with lysosomal marker LAMP-1 and blocking STING reduced the expression of cathepsin B and decreased the expression of Bax and caspase 3 cleavage. We observed similar protective effects after intranasal treatment with cGAS inhibitor RU.521, which were reversed by administration of STING agonist 2'3'-cGAMP. Additionally, we showed that long interspersed element 1 (LINE-1) retrotransposon, a potential upstream activator of cGAS/STING pathway was induced at 48 h after HI, which was evidenced by increased expression of ORF1p and ORF2p proteins and increased LINE-1 DNA content in the cytosol. Blocking LINE-1 with the nucleoside analog reverse-transcriptase inhibitor (NRTI) stavudine reduced infarction area, neuronal degeneration in the cerebral cortex, and reduced the expression of Bax and cleaved caspase 3. Thus, our results identify the cGAS/STING pathway as a potential therapeutic target to inhibit delayed neuronal death after HI.

Topics: Adaptor Proteins, Signal Transducing; Animals; bcl-2-Associated X Protein; Cathepsin B; Cell Death; Disease Models, Animal; Gene Silencing; Hypoxia-Ischemia, Brain; Long Interspersed Nucleotide Elements; Lysosomal Membrane Proteins; Membrane Proteins; Nerve Degeneration; Nucleotides, Cyclic; Nucleotidyltransferases; Rats; RNA, Small Interfering; Signal Transduction

Effectively activating macrophages against cancer is promising but challenging. In particular, cancer cells express CD47, a 'don't eat me' signal that interacts with signal regulatory protein alpha (SIRPα) on macrophages to prevent phagocytosis. Also, cancer cells secrete stimulating factors, which polarize tumor-associated macrophages from an antitumor M1 phenotype to a tumorigenic M2 phenotype. Here, we report that hybrid cell membrane nanovesicles (known as hNVs) displaying SIRPα variants with significantly increased affinity to CD47 and containing M2-to-M1 repolarization signals can disable both mechanisms. The hNVs block CD47-SIRPα signaling axis while promoting M2-to-M1 repolarization within tumor microenvironment, significantly preventing both local recurrence and distant metastasis in malignant melanoma models. Furthermore, by loading a stimulator of interferon genes (STING) agonist, hNVs lead to potent tumor inhibition in a poorly immunogenic triple negative breast cancer model. hNVs are safe, stable, drug loadable, and suitable for genetic editing. These properties, combined with the capabilities inherited from source cells, make hNVs an attractive immunotherapy.

Topics: Animals; CD47 Antigen; Cell Line, Tumor; Cell-Derived Microparticles; Disease Models, Animal; Female; HEK293 Cells; Humans; Immunotherapy; Macrophage Activation; Macrophages; Melanoma; Membrane Proteins; Mice; Nanoparticles; Neoplasm Recurrence, Local; Nucleotides, Cyclic; Receptors, Immunologic; Signal Transduction; Triple Negative Breast Neoplasms; Tumor Escape; Tumor Microenvironment

Topics: Adjuvants, Immunologic; Aging; Animals; Disease Models, Animal; Drug Delivery Systems; Female; Humans; Immunosenescence; Influenza A Virus, H1N1 Subtype; Influenza Vaccines; Mice; Mice, Inbred BALB C; Nucleotides, Cyclic; Orthomyxoviridae Infections; Saponins; Transdermal Patch

Reagents that activate the signaling adaptor stimulator of interferon genes (STING) suppress experimentally induced autoimmunity in murine models of multiple sclerosis and arthritis. In this study, we evaluated STING agonists as potential reagents to inhibit spontaneous autoimmune type I diabetes (T1D) onset in non-obese diabetic (NOD) female mice. Treatments with DNA nanoparticles (DNPs), which activate STING when cargo DNA is sensed, delayed T1D onset and reduced T1D incidence when administered before T1D onset. DNP treatment elevated indoleamine 2,3 dioxygenase (IDO) activity, which regulates T-cell immunity, in spleen, pancreatic lymph nodes and pancreas of NOD mice. Therapeutic responses to DNPs were partially reversed by inhibiting IDO and DNP treatment synergized with insulin therapy to further delay T1D onset and reduce T1D incidence. Treating pre-diabetic NOD mice with cyclic guanyl-adenyl dinucleotide (cGAMP) to activate STING directly delayed T1D onset and stimulated interferon-αβ (IFN-αβ), while treatment with cyclic diguanyl nucleotide (cdiGMP) did not delay T1D onset or induce IFN-αβ in NOD mice. DNA sequence analyses revealed that NOD mice possess a STING polymorphism that may explain differential responses to cGAMP and cdiGMP. In summary, STING agonists attenuate T1D progression and DNPs enhance therapeutic responses to insulin therapy.

Topics: Animals; Cyclic GMP; Diabetes Mellitus, Type 1; Disease Models, Animal; DNA; Drug Synergism; Female; Humans; Indoleamine-Pyrrole 2,3,-Dioxygenase; Insulin; Membrane Proteins; Mice; Mice, Inbred NOD; Nanoparticles; Nucleotides, Cyclic; Polymorphism, Genetic; T-Lymphocytes; Up-Regulation

The leading cause of mortality due to colorectal cancer (CRC) is highly associated with the development of liver metastases. Recently, we described cGAMP that is closely related to the metastatic state wherein the progress of metastatic tumors is associated with favorable outcomes in a zebrafish xenograft model. cGAMP was administered and the expression levels of type-I interferons were induced amongst tumor tissues to illuminate the overall measure of the induced STING/STAT3 axis in colorectal liver metastases. Furthermore, cGAMP-STING dependent STAT3 activation resulted in the inhibition of tumor cell proliferation, viability, and invasion in vitro. The subtotal reduction in tumor growth attributed to a large number of infiltrating inflammatory cells in vivo. We showed that cGAMP inhibited migration through angiogenesis by up-regulating IL-2, TNF-α, and IFN-γ, whereas STAT3 down-regulation inhibited CXCL8, BCL-2, and VEGFA expression. The importance of cGAMP in inhibiting the invasion front of CRC confirmed that the cGAMP dependent activation of STING/STAT3 axis played a key role in the inhibition of tumor progression.

Topics: Animals; Antineoplastic Agents; Colorectal Neoplasms; Disease Models, Animal; Heterografts; Liver Neoplasms; Membrane Proteins; Neoplasm Metastasis; Nucleotides, Cyclic; Signal Transduction; STAT3 Transcription Factor; Zebrafish; Zebrafish Proteins

Immunotherapies have significantly improved cancer patient survival, but response rates are still limited. Thus, novel formulations are needed to expand the breadth of immunotherapies. Pathogen associated molecular patterns (PAMPs) can be used to stimulate an immune response, but several pathogen recognition receptors are located within the cell, making delivery challenging. We have employed the biodegradable polymer acetalated dextran (Ace-DEX) to formulate PAMP microparticles (MPs) in order to enhance intracellular delivery. While treatment with four different PAMP MPs resulted in tumor growth inhibition, cyclic GMP-AMP (cGAMP) MPs were most effective. cGAMP MPs showed anti-tumor efficacy at doses 100-1000 fold lower than published doses of soluble cGAMP in two murine tumor models. Treatment with cGAMP MPs resulted in increased natural killer cell numbers in the tumor environment. Immune cell depletion studies confirmed that NK cells were responsible for the anti-tumor efficacy in an aggressive mouse melanoma model. NK cells and CD8

Topics: Acetylation; Animals; CD8-Positive T-Lymphocytes; Dextrans; Disease Models, Animal; Hydrodynamics; Immunity; Immunotherapy; Killer Cells, Natural; Melanoma; Membrane Proteins; Mice, Inbred C57BL; Microspheres; Neoplasms; Nucleotides, Cyclic; Pathogen-Associated Molecular Pattern Molecules; Triple Negative Breast Neoplasms; Tumor Burden

Pathogen recognition receptor (PRR) agonists are currently being developed and tested as adjuvants in various formulations to optimize the immunogenicity and efficacy of vaccines. Using an original in vitro approach to prime naive precursors from unfractionated human peripheral blood mononuclear cells, we assessed the influence of cyclic guanosine monophosphate-adenosine monophosphate (cGAMP), a ligand for the stimulator of interferon genes (STING), on the induction of antigen-specific CD8+ T cells. We found that 2'3'-cGAMP and 3'3'-cGAMP were especially potent adjuvants in this system, driving the expansion and maturation of functionally replete antigen-specific CD8+ T cells via the induction of type I IFNs. The biological relevance of these findings was confirmed in vivo using two mouse models, in which 2'3'-cGAMP-adjuvanted vaccination elicited protective antitumor or antiviral CD8+ T cell responses. These results identify particular isoforms of cGAMP as effective adjuvants that may find utility in the development of novel immunotherapies and vaccines.

Topics: Adjuvants, Immunologic; Animals; Cancer Vaccines; CD8-Positive T-Lymphocytes; Cell Line, Tumor; Cells, Cultured; Disease Models, Animal; Female; HIV Infections; HIV-1; Humans; Immunogenicity, Vaccine; Interferon Type I; Ligands; Membrane Proteins; Mice; Nucleotides, Cyclic; Primary Cell Culture; Thymoma; Thymus Neoplasms; Vaccination; Viral Vaccines

Innate immune responses are considered to play crucial roles in the progression of Alzheimer's disease (AD). Recently, immunotherapy is emerging as an innovative and highly conceivable strategy for AD treatment. The cGAMP-STING-IRF3 signaling pathway plays a pivotal role in mediating innate immune responses. In this study, we provide pioneering investigation to find that the STING stimulator, cGAMP, significantly ameliorates cognitive deficits, improves pathological changes, decreases Aβ plaque load and reduces neuron apoptosis in APP/PS1 transgenetic mice. The stimulation of cGAMP-STING-IRF3 pathway induces expression of triggering receptor expressed on myeloid cells 2 (TREM2), and the overexpression of TREM2 further decreases Aβ deposition and neuron loss while improves AD pathomorphology and cognitive impairment. Additionally, TREM2 regulates microglia polarization from M1 towards M2 phenotype thereby achieves reduction of neuroinflammation in AD. These findings support that the enhancement of TREM2 exerts beneficial effects in ameliorating AD development. Taken together, our results demonstrate that cGAMP is a potential candidate for applications in Alzheimer's disease immunotherapy.

Topics: Alzheimer Disease; Animals; Brain; Cognition Disorders; Cognitive Dysfunction; Disease Models, Animal; Disease Progression; Interferon Regulatory Factor-3; Male; Membrane Glycoproteins; Mice; Mice, Inbred C57BL; Mice, Transgenic; Microglia; Nucleotides, Cyclic; Phenotype; Receptors, Immunologic

Acute pancreatitis (AP) is characterized by severe inflammation and acinar cell death. Transmembrane protein 173 (TMEM173 or STING) is a DNA sensor adaptor protein on immune cells that recognizes cytosolic nucleic acids and transmits signals that activate production of interferons and the innate immune response. We investigated whether leukocyte STING signaling mediates inflammation in mice with AP.. We induced AP in C57BL/6J mice (control) and C57BL/6J-Tmem173gt/J mice (STING-knockout mice) by injection of cerulein or placement on choline-deficient DL-ethionine supplemented diet. In some mice, STING signaling was induced by administration of a pharmacologic agonist. AP was also induced in C57BL/6J mice with bone marrow transplants from control or STING-knockout mice and in mice with disruption of the cyclic GMP-AMP synthase (Cgas) gene. Pancreata were collected, analyzed by histology, and acini were isolated and analyzed by flow cytometry, quantitative polymerase chain reaction, immunoblots, and enzyme-linked immunosorbent assay. Bone-marrow-derived macrophages were collected from mice and tested for their ability to detect DNA from dying acinar cells in the presence and absence of deoxyribonuclease (DNaseI).. STING signaling was activated in pancreata from mice with AP but not mice without AP. STING-knockout mice developed less severe AP (less edema, inflammation, and markers of pancreatic injury) than control mice, whereas mice given a STING agonist developed more severe AP than controls. In immune cells collected from pancreata, STING was expressed predominantly in macrophages. Levels of cGAS were increased in mice with vs without AP, and cGAS-knockout mice had decreased edema, inflammation, and other markers of pancreatic injury upon induction of AP than control mice. Wild-type mice given bone marrow transplants from STING-knockout mice had less pancreatic injury and lower serum levels of lipase and pancreatic trypsin activity following induction of AP than mice given wild-type bone marrow. DNA from dying acinar cells activated STING signaling in macrophages, which was inhibited by addition of DNaseI.. In mice with AP, STING senses acinar cell death (by detecting DNA from dying acinar cells) and activates a signaling pathway that promotes inflammation. Macrophages express STING and activate pancreatic inflammation in AP.

Topics: Acinar Cells; Acute Disease; Animals; Cell Death; Ceruletide; Disease Models, Animal; Inflammation; Macrophages; Membrane Proteins; Mice; Mice, Inbred C57BL; Mice, Knockout; Nucleotides, Cyclic; Pancreas; Pancreatitis; Signal Transduction

Detection of intracellular DNA triggers activation of the stimulator of IFN genes-dependent IFN-stimulatory DNA (ISD) pathway, which is essential for antiviral immune responses. However, chronic activation of this pathway is implicated in autoimmunity. Mutations in TREX1, a 3' repair exonuclease that degrades cytosolic DNA, cause Aicardi-Goutières syndrome and chilblain lupus. Trex1 (-/-) mice develop lethal, IFN-driven autoimmune disease that is dependent on activation of the ISD pathway, but the DNA sensors that detect the endogenous DNA that accumulates in Trex1 (-/-) mice have not been defined. Multiple DNA sensors have been proposed to activate the ISD pathway, including cyclic GMP-AMP synthase (cGAS). In this study, we show that Trex1 (-/-) mice lacking cGAS are completely protected from lethality, exhibit dramatically reduced tissue inflammation, and fail to develop autoantibodies. These findings implicate cGAS as a key driver of autoimmune disease and suggest that cGAS inhibitors may be useful therapeutics for Aicardi-Goutières syndrome and related autoimmune diseases.

Topics: Animals; Autoantibodies; Autoimmune Diseases of the Nervous System; Cells, Cultured; Disease Models, Animal; Embryo, Mammalian; Exodeoxyribonucleases; Fibroblasts; Gene Expression; Humans; Immunoblotting; Inflammation; Interferon-beta; Interferons; Macrophages; Mice, Inbred C57BL; Mice, Knockout; Nervous System Malformations; Nucleotides, Cyclic; Nucleotidyltransferases; Phosphoproteins; Reverse Transcriptase Polymerase Chain Reaction

Spontaneous CD8 T-cell responses occur in growing tumors but are usually poorly effective. Understanding the molecular and cellular mechanisms that drive these responses is of major interest as they could be exploited to generate a more efficacious antitumor immunity. As such, stimulator of IFN genes (STING), an adaptor molecule involved in cytosolic DNA sensing, is required for the induction of antitumor CD8 T responses in mouse models of cancer. Here, we find that enforced activation of STING by intratumoral injection of cyclic dinucleotide GMP-AMP (cGAMP), potently enhanced antitumor CD8 T responses leading to growth control of injected and contralateral tumors in mouse models of melanoma and colon cancer. The ability of cGAMP to trigger antitumor immunity was further enhanced by the blockade of both PD1 and CTLA4. The STING-dependent antitumor immunity, either induced spontaneously in growing tumors or induced by intratumoral cGAMP injection was dependent on type I IFNs produced in the tumor microenvironment. In response to cGAMP injection, both in the mouse melanoma model and an ex vivo model of cultured human melanoma explants, the principal source of type I IFN was not dendritic cells, but instead endothelial cells. Similarly, endothelial cells but not dendritic cells were found to be the principal source of spontaneously induced type I IFNs in growing tumors. These data identify an unexpected role of the tumor vasculature in the initiation of CD8 T-cell antitumor immunity and demonstrate that tumor endothelial cells can be targeted for immunotherapy of melanoma.

Topics: Animals; Antigens, Neoplasm; CD8-Positive T-Lymphocytes; Cell Proliferation; CTLA-4 Antigen; Dendritic Cells; Disease Models, Animal; Dose-Response Relationship, Immunologic; Endothelial Cells; Immunity; Injections, Intralesional; Interferon Type I; Lymphocytes, Tumor-Infiltrating; Melanoma; Melanoma, Experimental; Membrane Proteins; Mice, Inbred C57BL; Neoplasms; Nucleotides, Cyclic; Receptor, Interferon alpha-beta; Signal Transduction