Cytosolic Nucleic Acid Sensors Research Articles

Abstract 5617: Combined inhibition of ATR and ATM with tuvusertib and lartesertib (M4076) impacts the tumor microenvironment

Abstract Ataxia telangiectasia and Rad3-related protein (ATR) and ataxia telangiectasia mutated (ATM) are two of the main kinases of the DNA damage response (DDR). Recent work has demonstrated that the addition of ATM inhibitors enhances the cytotoxicity and in vivo anti-tumor efficacy of ATR inhibitors by abrogating the ATR inhibitor mediated G1 cell cycle arrest and enhancing chromosomal damage.1 Prior to causing cell death, damaged chromosome fragments can end up in the cytosol where they are detected by cytosolic nucleic acid sensors such as cGAS, resulting in the induction of inflammatory signaling pathways and an anti-tumor immune response. This suggests that the mode of action of DDR inhibitors may involve the tumor microenvironment (TME). We therefore aimed to assess the influence of combined pharmacological ATR and ATM inhibition on the TME to better understand how this could be exploited therapeutically. In line with published results in human tumor models, the treatment of murine colon cancer MC-38 cells in vitro with a combination of ATR inhibitor tuvusertib and ATM inhibitor lartesertib led to cell death. Further analysis revealed an activation of the cGAS-STING signaling pathway, an upregulation of PD-L1 and the release of inflammatory cytokines. These results confirmed the activation of immune signaling pathways upon combined inhibition of ATR and ATM. To analyze the stimulation of an anti-tumor immune response upon tuvusertib + lartesertib treatment in vivo, MC-38 cells were transplanted into immunocompetent C57BL/6J mice. Immunoprofiling by flow cytometry and immunohistochemistry staining confirmed that treatment with tuvusertib + lartesertib indeed altered the TME in this setting. In line with the in vitro findings, a significant upregulation of PD-L1 on immune and tumor cells could be observed after 24, 48, and 72 h of treatment even though MC-38 cells expressed high levels of PD-L1 already. Immunoprofiling did not reveal any changes in the TME immune cell content at these early time points. Interestingly, PD-L1 expression as well as tumor immune population changed after prolonged (23 days) treatment. PD-L1 was still expressed on tumor cells but to a significantly lesser extent and a depletion of CD8+ T-cells was observed. However, a significant increase in NK cells could be detected. In summary, we show that inhibition of the DDR through a combination of ATR and ATM inhibitors influences the TME in the MC-38 tumor model. These findings suggest an additional mode of action beyond direct cancer cell cytotoxicity. Further studies on how this may be leveraged therapeutically, for example through a combination with immune checkpoint inhibitors, are warranted. 1. Turchick A, Zimmermann A, et al. Mol Cancer Ther 2023;22(7):859-872. Citation Format: Karin Laaber, Audrey Turchick, Julia Jabs, Brian Elenbaas, Lyubomir T. Vassilev, Astrid Zimmermann. Combined inhibition of ATR and ATM with tuvusertib and lartesertib (M4076) impacts the tumor microenvironment [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 1 (Regular Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(6_Suppl):Abstract nr 5617.

The poxvirus F17 protein counteracts mitochondrially orchestrated antiviral responses

Poxviruses are unusual DNA viruses that replicate in the cytoplasm. To do so, they encode approximately 100 immunomodulatory proteins that counteract cytosolic nucleic acid sensors such as cGAMP synthase (cGAS) along with several other antiviral response pathways. Yet most of these immunomodulators are expressed very early in infection while many are variable host range determinants, and significant gaps remain in our understanding of poxvirus sensing and evasion strategies. Here, we show that after infection is established, subsequent progression of the viral lifecycle is sensed through specific changes to mitochondria that coordinate distinct aspects of the antiviral response. Unlike other viruses that cause extensive mitochondrial damage, poxviruses sustain key mitochondrial functions including membrane potential and respiration while reducing reactive oxygen species that drive inflammation. However, poxvirus replication induces mitochondrial hyperfusion that independently controls the release of mitochondrial DNA (mtDNA) to prime nucleic acid sensors and enables an increase in glycolysis that is necessary to support interferon stimulated gene (ISG) production. To counter this, the poxvirus F17 protein localizes to mitochondria and dysregulates mTOR to simultaneously destabilize cGAS and block increases in glycolysis. Our findings reveal how the poxvirus F17 protein disarms specific mitochondrially orchestrated responses to later stages of poxvirus replication.

Spatiotemporal expression patterns of ZBP1 in the brain of mouse experimental stroke model

Z-DNA binding protein 1 (ZBP1) is a cytosolic nucleic acid sensor, functioning as a critical mediator of inflammation and cell death pathways. Since neuroinflammation could occur in response to damage-associated molecular patterns (DAMPs), ZBP1 might be involved in neuroinflammation after stroke. However, the spatiotemporal expression profile of ZBP1 in the post-stroke brain remains to be elucidated. The aim of this study is to demonstrate the spatiotemporal expression patterns of ZBP1 in the post-stroke brain using a mouse photothrombotic stroke model. Real-time PCR assays showed that ZBP1 is induced on days 3–14 post stroke. ZBP1 immunoreactivity was observed in Iba1-positive microglia/macrophages in peri-infarct regions by immunohistochemistry. ZBP1-positive cells were spread in layers surrounding the infarct core by 7–14 days post stroke. Interestingly, ZBP1 immunoreactivity was also detected in CD206-positive border-associated macrophages (BAMs) in the meninges. Furthermore, ZBP1-expressing cells were positive for antibodies against inflammatory mediators such as Toll-like receptor 4 (TLR4), Toll/IL-1R domain-containing adaptor-inducing IFN-β (TRIF), and receptor-interacting serine/threonine-protein kinase 1 (RIPK1). Morphological analysis with confocal microscopy showed that the co-localization signals of ZBP1 and its adaptor, TRIF, are increased by glucose oxidase (GOx) treatment, which has been reported to induce mitochondrial DNA (mtDNA) release. These results suggest that ZBP1 is induced in peri-infarct microglia/macrophages and may be involved in DAMPs-mediated neuroinflammation involving mtDNA in the post-infarct brain.

Cooperative sensing of mitochondrial DNA by ZBP1 and cGAS promotes cardiotoxicity

Mitochondrial DNA (mtDNA) is a potent agonist of the innate immune system; however, the exact immunostimulatory features of mtDNA and the kinetics of detection by cytosolic nucleic acid sensors remain poorly defined. Here, we show that mitochondrial genome instability promotes Z-form DNA accumulation. Z-DNA binding protein 1 (ZBP1) stabilizes Z-form mtDNA and nucleates a cytosolic complex containing cGAS, RIPK1, and RIPK3 to sustain STAT1 phosphorylation and type I interferon (IFN-I) signaling. Elevated Z-form mtDNA, ZBP1 expression, and IFN-I signaling are observed in cardiomyocytes after exposure to Doxorubicin, a first-line chemotherapeutic agent that induces frequent cardiotoxicity in cancer patients. Strikingly, mice lacking ZBP1 or IFN-I signaling are protected from Doxorubicin-induced cardiotoxicity. Our findings reveal ZBP1 as a cooperative partner for cGAS that sustains IFN-I responses to mitochondrial genome instability and highlight ZBP1 as a potential target in heart failure and other disorders where mtDNA stress contributes to interferon-related pathology.

Human ZBP1 induces cell death-independent inflammatory signaling via RIPK3 and RIPK1.

ZBP1 is an interferon-induced cytosolic nucleic acid sensor that facilitates antiviral responses via RIPK3. Although ZBP1-mediated programmed cell death is widely described, whether and how it promotes inflammatory signaling is unclear. Here, we report a ZBP1-induced inflammatory signaling pathway mediated by K63- and M1-linked ubiquitin chains, which depends on RIPK1 and RIPK3 as scaffolds independently of cell death. In human HT29 cells, ZBP1 associated with RIPK1 and RIPK3 as well as ubiquitin ligases cIAP1 and LUBAC. ZBP1-induced K63- and M1-linked ubiquitination of RIPK1 and ZBP1 to promote TAK1- and IKK-mediated inflammatory signaling and cytokine production. Inhibition of caspase activity suppressed ZBP1-induced cell death but enhanced cytokine production in a RIPK1- and RIPK3 kinase activity-dependent manner. Lastly, we provide evidence that ZBP1 signaling contributes to SARS-CoV-2-induced cytokine production. Taken together, we describe a ZBP1-RIPK3-RIPK1-mediated inflammatory signaling pathway relayed by the scaffolding role of RIPKs and regulated by caspases, which may induce inflammation when ZBP1 is activated below the threshold needed to trigger a cell death response.

Inherited IRAK-4 Deficiency in Acute Human Herpesvirus-6 Encephalitis.

Human herpesvirus-6 (HHV-6) infection can rarely cause life-threatening conditions, such as encephalitis, in otherwise healthy children, with unclear pathogenesis. We studied a child who presented with acute HHV-6 encephalitis at the age of 10 months and who was homozygous for a novel missense mutation in IRAK4, encoding interleukin-1 receptor-associated kinase 4, identified by whole-exome sequencing. We tested the damaging impact of this mutation in silico by molecular dynamics simulations and in vitro by biochemical and functional experiments utilizing cell lines and patient’s cells. We found that the mutation is severely hypomorphic, impairing both the expression and function of IRAK-4. Patient’s leukocytes had barely detectable levels of IRAK-4 and diminished anti-viral immune responses to various stimuli inducing different Toll-like receptors and cytosolic nucleic acid sensors. Overall, these findings suggest that acute HHV-6 encephalitis can result from inborn errors of immunity to virus. This study represents the first report of isolated acute HHV-6 infection causing encephalitis in an inherited primary immunodeficiency, notably autosomal recessive (AR) partial IRAK-4 deficiency, and the first report of AR IRAK-4 deficiency presenting with a severe viral disease, notably HHV-6 encephalitis upon an acute infection, thereby expanding the clinical spectrum of IRAK-4 deficiency.Supplementary InformationThe online version contains supplementary material available at 10.1007/s10875-022-01369-4.

RNA-Editing Enzyme ADAR1 p150 Isoform Is Critical for Germinal Center B Cell Response.

Adenosine deaminase acting on RNA (ADAR)1 is the principal enzyme for adenosine-to-inosine editing, an RNA modification-avoiding cytosolic nucleic acid sensor's activation triggered by endogenous dsRNAs. Two ADAR1 isoforms exist in mammals, a longer IFN-inducible and mainly cytoplasm-localized p150 isoform and a shorter constitutively expressed and primarily nucleus-localized p110 isoform. Studies of ADAR1 mutant mice have demonstrated that ADAR1 is essential for multiple physiological processes, including embryonic development, innate immune response, and B and T lymphocyte development. However, it remained unknown whether ADAR1 plays a role in the humoral immune response. In this study, we conditionally delete Adar1 in activated B cells and show that ADAR1-deficient mice have a defective T cell-dependent Ab response and diminished germinal center (GC) B cells. Using various double mutant mice concurrently deficient in ADAR1 and different downstream dsRNA sensors, we demonstrate that ADAR1 regulates the GC response by preventing hyperactivation of the melanoma differentiation-associated protein 5 (MDA5) but not the protein kinase R or RNase L pathway. We also show that p150 is exclusively responsible for ADAR1's function in the GC response, and the p110 isoform cannot substitute for the p150's role, even when p110 is constitutively expressed in the cytoplasm. We further demonstrated that the dsRNA-binding but not the RNA-editing activity is required for ADAR1's function in the GC response. Thus, our data suggest that the ADAR1 p150 isoform plays a crucial role in regulating the GC B cell response.

Post-Translational Modifications of Proteins in Cytosolic Nucleic Acid Sensing Signaling Pathways.

The innate immune response is the first-line host defense against pathogens. Cytosolic nucleic acids, including both DNA and RNA, represent a special type of danger signal to initiate an innate immune response. Activation of cytosolic nucleic acid sensors is tightly controlled in order to achieve the high sensitivity needed to combat infection while simultaneously preventing false activation that leads to pathologic inflammatory diseases. In this review, we focus on post-translational modifications of key cytosolic nucleic acid sensors that can reversibly or irreversibly control these sensor functions. We will describe phosphorylation, ubiquitination, SUMOylation, neddylation, acetylation, methylation, succinylation, glutamylation, amidation, palmitoylation, and oxidation modifications events (including modified residues, modifying enzymes, and modification function). Together, these post-translational regulatory modifications on key cytosolic DNA/RNA sensing pathway members reveal a complicated yet elegantly controlled multilayer regulator network to govern innate immune activation.

Abstract 3205: Role of RIG-I in tumor endothelium

Abstract The immune microenvironment plays a critical role in the efficacy of cancer treatment. The manipulation of cytosolic nucleic acid sensors has become a strategy to boost the immune response in cancer therapy, but it is critical to understand how these nucleic acid sensors work in the tumor microenvironment. One aspect of the immune microenvironment is the nucleic acid sensor, which recognizes pathogenic nucleic acids and activates an immune response. Retinoic acid-inducible gene I (RIG-I) is an RNA sensor that responds to dsRNA by activating a type I interferon response. It has been found that RIG-I activation can induce an anti-tumor immune response, but its role in the tumor microenvironment is unknown. Prior studies show regulation of the innate immune response in endothelium can increase the anti-tumor response. Understanding the role of RIG-I in endothelial cells, and how this can be used to potentially reprogram the tumor microenvironment, is critical for developing more effective cancer treatments. In addition, my research on the effects of RIG-I in endothelium has wide-reaching implications beyond cancer treatment. I hypothesize RIG-I activation in endothelial cells will produce an anti-tumor immune response. My findings show that endothelial RIG-I activates a robust type I interferon response and inhibits EC function including, inducing apoptosis and inhibiting migration, which is indicative that RIG-I plays a role in the endothelial immune response. RIG-I also plays a role in the expression of the several angiogenic regulators in a tissue-specific manner. This research will provide critical knowledge about the role of RIG-I in tumor angiogenesis, and provide novel treatment avenues for solid tumors. Citation Format: Adrian M. Baris, Eugenia Fraile-Bethencourt, Sokchea Khou, Rebecca Ruhl, Clay Hudson, Sudarshan Anand. Role of RIG-I in tumor endothelium [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 3205.

Z-DNA binding protein 1 mediates necroptotic and apoptotic cell death pathways in murine astrocytes following herpes simplex virus-1 infection

BackgroundThe mechanisms by which glia respond to viral central nervous system (CNS) pathogens are now becoming apparent with the demonstration that microglia and astrocytes express an array of pattern recognition receptors that include intracellular RNA and DNA sensors. We have previously demonstrated that glia express Z-DNA binding protein 1 (ZBP1) and showed that this cytosolic nucleic acid sensor contributes to the inflammatory/neurotoxic responses of these cells to herpes simplex virus-1 (HSV-1). However, the relative contribution made by ZBP1- to HSV-1-mediated cell death in glia has not been determined.MethodsWe have investigated the relative contribution made by ZBP1- to HSV-1-mediated cell death in primary astrocytes derived from mice genetically deficient in this sensor. We have used capture ELISAs and immunoblot analysis to assess inflammatory cytokine production and ZBP1 and phosphorylated mixed lineage kinase domain-like protein (MLKL) expression levels, respectively, following HSV-1 challenge. Furthermore, we have used a commercially available cell viability assay to determine the proportion and rate of cell death in cells following infection with laboratory and neuroinvasive clinical strains of HSV-1, and pharmacological inhibitors of necroptotic and apoptotic pathway components to assess the relative role of each.ResultsWe show that the loss of ZBP1 in astrocytes results in an increase in the number of viral particles released following HSV-1 infection. Importantly, we have confirmed that HSV-1 induces necroptosis in astrocytes and have established the ability of ZBP1 to mediate this cell death pathway. Interestingly, while ZBP1 is best known for its role in necroptotic signaling, our findings indicate that this sensor can also contribute to virally induced apoptosis in these glia.ConclusionsOur findings indicate that ZBP1 serves as a restriction factor for HSV-1 infection and is associated with the induction of both necroptotic and apoptotic cell death pathways in primary murine astrocytes. While it remains to be seen whether ZBP1-mediated activation of cell death in astrocytes contributes significantly to host protection or, rather, exacerbates HSV-1 encephalitis pathology, the identification of such a role in resident CNS cells may represent a novel target for therapeutic intervention to reduce HSV encephalitis-associated morbidity and mortality.

Role of RIG-I in tumor endothelium.

Abstract The immune microenvironment plays a critical role in the efficacy of cancer treatment. The manipulation of cytosolic nucleic acid sensors has become a strategy to boost the immune response in cancer therapy, but it is critical to understand how these nucleic acid sensors work in the tumor microenvironment. One aspect of the immune microenvironment is the nucleic acid sensor, which recognizes pathogenic nucleic acids and activates an immune response. Retinoic acid-inducible gene I (RIG-I) is an RNA sensor that responds to dsRNA by activating a type I interferon response. It has been found that RIG-I activation can induce an anti-tumor immune response, but its role in the tumor microenvironment is unknown. Prior studies show regulation of the innate immune response in endothelium can increase the anti-tumor response. Understanding the role of RIG-I in endothelial cells, and how this can be used to potentially reprogram the tumor microenvironment, is critical for developing more effective cancer treatments. In addition, my research on the effects of RIG-I in endothelium has wide-reaching implications beyond cancer treatment. I hypothesize RIG-I activation in endothelial cells will produce an anti-tumor immune response. My findings show that endothelial RIG-I activates a robust type I interferon response and inhibits EC function including, inducing apoptosis and inhibiting migration, which is indicative that RIG-I plays a role in the endothelial immune response. RIG-I also plays a role in the expression of the several angiogenic regulators in a tissue-specific manner. This research will provide critical knowledge about the role of RIG-I in tumor angiogenesis, and provide novel treatment avenues for solid tumors. Supported by grants from NIH (T32) and Knight (CVP-002)

The crosstalk between the caspase family and the cGAS‒STING signaling pathway.

Edited by Jiarui Wu Cytosolic nucleic acid sensors are critical for sensing nucleic acids and initiating innate immunity during microbial infections and/or cell death. Over the last decade, several key studies have characterized the conserved mechanism of cyclic guanosine monophosphate‒adenosine monophosphate synthase (cGAS) and the downstream signaling adaptor stimulator of interferon genes (STING) initiating the innate immune signaling pathways. Aside from its primary involvement in microbial infections and inflammatory diseases, there is growing interest in the alternate roles of cGAS‒STING-mediated signaling. Caspase family members are powerful functional proteins that respond to cellular stress, including cell death signals, inflammation, and innate immunity. Recent studies have uncovered how the caspase family cooperates with the cGAS‒STING signaling pathway. Most caspase family members negatively regulate the cGAS‒STING signaling pathway. In turn, some caspase family members can also be modulated by cGAS‒STING. This review gives a detailed account of the interplay between the caspase family and the cGAS‒STING signaling pathway, which will shed light on developing novel therapeutics targeting the caspase family and cGAS‒STING signaling in antiviral innate immunity, cancer, inflammatory, and autoimmunity.

Identification of a Novel cGas/STING/Type I Interferon Specific Murine Cardiac Immune Landscape Following Radiation Therapy

<h3>Purpose/Objective(s)</h3> Cardiotoxicity from radiation treatment to the chest is a late toxicity that has been shown to increase with radiation dose. While clinical parameters and advances in radiation techniques have resulted in lower doses to the heart, the pathophysiologic mechanisms of radiation cardiotoxicity remains a debated topic. We have previously demonstrated that, in murine models, radiation activates a delayed, fibroblast-specific type I interferon response that is dependent on the cytosolic nucleic acid sensor, cGas and its downstream signaling protein STING. Indeed, STING knockout mice exhibit a survival advantage in comparison to wild type mice following a radiation challenge to the heart (20Gy x 1). In this study, we sought to evaluate the consequences of the type I interferon activation on cardiac tissue-specific white blood cell population and identify novel radiation-dependent changes of cardiac immune infiltrates. <h3>Materials/Methods</h3> All animal experiments were performed in accordance with IACUC guidelines. C57BL/6 wild type mice and C57BL/6 Tmem173^gt (catalytically inactive STING) were treated with either sham or 12 Gy single dose to the heart using opposing tangents. At 1 month, hearts were harvested and CD45+ positive cells were immunoprecipitated and sent for 3' single cell RNA (scRNA) sequencing using 10X genomics. ScRNA data was analyzed using Seurat 3.0 <h3>Results</h3> Our results show that radiation induces the upregulation of a subset of cardiac macrophages, interferon inducible cardiac (IFNIC) macrophages. This effect is that is abrogated in mice lacking a functional STING. Furthermore, we observed a general activation of innate immunity as evidenced by increases in the dendritic cell population as well as upregulation of non-classical class I MHC molecules in non-IFNIC macrophages. More surprisingly, we find that there is a marked increase in tissue specific B cell population following radiation, an effect that is completely abolished in mice lacking a functional STING. <h3>Conclusion</h3> We describe for the first time the immune landscape of heart tissue following radiation. We find a previously undescribed infiltration of IFNIC macrophages and a robust activation of an innate immunity. Furthermore, we find a strong effector humoral adaptive response, likely downstream. These findings open the possibility to further understand the role of immune infiltrates in mediation of normal tissue toxicity following radiation.

Increased presence of nuclear DNAJA3 and upregulation of cytosolic STAT1 and of nucleic acid sensors trigger innate immunity in the ClpP-null mouse

Mitochondrial dysfunction may activate innate immunity, e.g. upon abnormal handling of mitochondrial DNA in TFAM mutants or in altered mitophagy. Recent reports showed that also deletion of mitochondrial matrix peptidase ClpP in mice triggers transcriptional upregulation of inflammatory factors. Here, we studied ClpP-null mouse brain at two ages and mouse embryonal fibroblasts, to identify which signaling pathways are responsible, employing mass spectrometry, subcellular fractionation, immunoblots, and reverse transcriptase polymerase chain reaction. Several mitochondrial unfolded protein response factors showed accumulation and altered migration in blue-native gels, prominently the co-chaperone DNAJA3. Its mitochondrial dysregulation increased also its extra-mitochondrial abundance in the nucleus, a relevant observation given that DNAJA3 modulates innate immunity. Similar observations were made for STAT1, a putative DNAJA3 interactor. Elevated expression was observed not only for the transcription factors Stat1/2, but also for two interferon-stimulated genes (Ifi44, Gbp3). Inflammatory responses were strongest for the RLR pattern recognition receptors (Ddx58, Ifih1, Oasl2, Trim25) and several cytosolic nucleic acid sensors (Ifit1, Ifit3, Oas1b, Ifi204, Mnda). The consistent dysregulation of these factors from an early age might influence also human Perrault syndrome, where ClpP loss-of-function leads to early infertility and deafness, with subsequent widespread neurodegeneration.

Cyclic Guanosine Monophosphate-Adenosine Monophosphate Synthase (cGAS), a Multifaceted Platform of Intracellular DNA Sensing.

Innate immune pathways are the first line of cellular defense against pathogen infections ranging from bacteria to Metazoa. These pathways are activated following the recognition of pathogen associated molecular patterns (PAMPs) by membrane and cytosolic pattern recognition receptors. In addition, some of these cellular sensors can also recognize endogenous danger-associated molecular patterns (DAMPs) arising from damaged or dying cells and triggering innate immune responses. Among the cytosolic nucleic acid sensors, the cyclic guanosine monophosphate–adenosine monophosphate (cGAMP) synthase (cGAS) plays an essential role in the activation of the type I interferon (IFNs) response and the production of pro-inflammatory cytokines. Indeed, upon nucleic acid binding, cGAS synthesizes cGAMP, a second messenger mediating the activation of the STING signaling pathway. The functional conservation of the cGAS-STING pathway during evolution highlights its importance in host cellular surveillance against pathogen infections. Apart from their functions in immunity, cGAS and STING also play major roles in nuclear functions and tumor development. Therefore, cGAS-STING is now considered as an attractive target to identify novel biomarkers and design therapeutics for auto-inflammatory and autoimmune disorders as well as infectious diseases and cancer. Here, we review the current knowledge about the structure of cGAS and the evolution from bacteria to Metazoa and present its main functions in defense against pathogens and cancer, in connection with STING. The advantages and limitations of in vivo models relevant for studying the cGAS-STING pathway will be discussed for the notion of species specificity and in the context of their integration into therapeutic screening assays targeting cGAG and/or STING.

CGAS- Stimulator of Interferon Genes Signaling in Central Nervous System Disorders.

Cytosolic nucleic acid sensors contribute to the initiation of innate immune responses by playing a critical role in the detection of pathogens and endogenous nucleic acids. The cytosolic DNA sensor cyclic-GMP-AMP synthase (cGAS) and its downstream effector, stimulator of interferon genes (STING), mediate innate immune signaling by promoting the release of type I interferons (IFNs) and other inflammatory cytokines. These biomolecules are suggested to play critical roles in host defense, senescence, and tumor immunity. Recent studies have demonstrated that cGAS-STING signaling is strongly implicated in the pathogenesis of central nervous system (CNS) diseases which are underscored by neuroinflammatory-driven disease progression. Understanding and regulating the interactions between cGAS-STING signaling and the nervous system may thus provide an effective approach to prevent or delay late-onset CNS disorders. Here, we present a review of recent advances in the literature on cGAS-STING signaling and provide a comprehensive overview of the modulatory patterns of the cGAS-STING pathway in CNS disorders.

Crosstalk between cGAS-STING signaling and cell death.

Cytosolic nucleic acid sensors have a critical role in detecting endogenous nucleic acids to initiate innate immune responses during microbial infections and/or cell death. Several seminal studies over the past decade have delineated the conserved mechanism of cytosolic DNA sensor cyclic GMP-AMP synthase (cGAS) andthe downstream signaling adapter stimulator of interferon genes (STING) in mediating innate immune signaling pathways as a host defense mechanism. Besides the predominant role in microbial infections and inflammatory diseases, there is an increased attention on alternative functional responses of cGAS-STING-mediated signaling. Here we review the complexity of interactions between the cGAS-STING signaling and cell death pathways. A better understanding of molecular mechanisms of this interplay is important with regard to the development of new therapeutics targeting cGAS-STING signaling in cancer, infectious, and chronic inflammatory diseases.

ABCF1 Regulates dsDNA-induced Immune Responses in Human Airway Epithelial Cells.

Background: The airway epithelium represents a critical component of the human lung that helps orchestrate defenses against respiratory tract viral infections, which are responsible for more than 2.5 million deaths/year globally. Innate immune activities of the airway epithelium rely on Toll-like receptors (TLRs), nucleotide binding and leucine-rich-repeat pyrin domain containing (NLRP) receptors, and cytosolic nucleic acid sensors. ATP Binding Cassette (ABC) transporters are ubiquitous across all three domains of life—Archaea, Bacteria, and Eukarya—and expressed in the human airway epithelium. ABCF1, a unique ABC family member that lacks a transmembrane domain, has been defined as a cytosolic nucleic acid sensor that regulates CXCL10, interferon-β expression, and downstream type I interferon responses. We tested the hypothesis that ABCF1 functions as a dsDNA nucleic acid sensor in human airway epithelial cells important in regulating antiviral responses.Methods: Expression and localization experiments were performed using in situ hybridization and immunohistochemistry in human lung tissue, while confirmatory transcript and protein expression was performed in human airway epithelial cells. Functional experiments were performed with siRNA methods in a human airway epithelial cell line. Complementary transcriptomic analyses were performed to explore the contributions of ABCF1 to gene expression patterns.Results: Using archived human lung and human airway epithelial cells, we confirm expression of ABCF1 gene and protein expression in these tissue samples, with a role for mediating CXCL10 production in response to dsDNA viral mimic challenge. Although, ABCF1 knockdown was associated with an attenuation of select genes involved in the antiviral responses, Gene Ontology analyses revealed a greater interaction of ABCF1 with TLR signaling suggesting a multifactorial role for ABCF1 in innate immunity in human airway epithelial cells.Conclusion: ABCF1 is a candidate cytosolic nucleic acid sensor and modulator of TLR signaling that is expressed at gene and protein levels in human airway epithelial cells. The precise level where ABCF1 protein functions to modulate immune responses to pathogens remains to be determined but is anticipated to involve IRF-3 and CXCL10 production.

Dectin-1 Promotes Type I and III Interferon Expression to Support Optimal Antifungal Immunity in the Lung.

Pulmonary infections with Aspergillus fumigatus (Af) are a significant cause of invasive fungal disease and lead to high morbidity and mortality in diverse populations throughout the world. Currently available antifungal drugs are often ineffective, thus contributing to unacceptably high mortality rates in patients suffering from invasive fungal infections. The use of cytokines as adjunctive immune therapies holds the promise of significantly improving patient outcomes in the future. In recent studies, we identified an essential role for type I and III interferons as regulators of optimal antifungal responses by pulmonary neutrophils during infection with Af. Although various membrane and cytosolic nucleic acid sensors are known to regulate interferon production in response to viruses, the pathways that regulate the production of these cytokines during fungal infection remain uncovered. In the current study, we demonstrate that dectin-1-mediated recognition of β-glucan on the cell wall of the clinically relevant fungal pathogen Aspergillus fumigatus promotes the activation of a protective cascade of type I and III interferon expression. We further demonstrate that exogenous administration of type I and III interferons can rescue inadequate antifungal responses in dectin-1−/− mice, suggesting the potential therapeutic benefit of these cytokines as activators of antifungal defense in the context of innate defects.

Inflammatory microenvironment remodelling by tumour cells after radiotherapy

The development of immune checkpoint inhibitors (ICIs) is revolutionizing the way we think about cancer treatment. Even so, for most types of cancer, only a minority of patients currently benefit from ICI therapies. Intrinsic and acquired resistance to ICIs has focused research towards new combination therapy approaches that seek to increase response rates, the depth of remission and the durability of benefit. In this Review, we describe how radiotherapy, through its immunomodulating effects, represents a promising combination partner with ICIs. We describe how recent research on DNA damage response (DDR) inhibitors in combination with radiotherapy may be used to augment this approach. Radiotherapy can kill cancer cells while simultaneously triggering the release of pro-inflammatory mediators and increasing tumour-infiltrating immune cells - phenomena often described colloquially as turning immunologically 'cold' tumours 'hot'. Here, we focus on new developments illustrating the key role of tumour cell-autonomous signalling after radiotherapy. Radiotherapy-induced tumour cell micronuclei activate cytosolic nucleic acid sensor pathways, such as cyclic GMP-AMP synthase (cGAS)-stimulator of interferon genes (STING), and propagation of the resulting inflammatory signals remodels the immune contexture of the tumour microenvironment. In parallel, radiation can impact immunosurveillance by modulating neoantigen expression. Finally, we highlight how tumour cell-autonomous mechanisms might be exploited by combining DDR inhibitors, ICIs and radiotherapy.