Sensor Stimulator Of Interferon Genes Research Articles

Myocardial Mitochondrial DNA Drives Macrophage Inflammatory Response through STING Signaling in Coxsackievirus B3-Induced Viral Myocarditis.

Coxsackievirus B3 (CVB3), a single-stranded positive RNA virus, primarily infects cardiac myocytes and is a major causative pathogen for viral myocarditis (VMC), driving cardiac inflammation and organ dysfunction. However, whether and how myocardial damage is involved in CVB3-induced VMC remains unclear. Herein, we demonstrate that the CVB3 infection of cardiac myocytes results in the release of mitochondrial DNA (mtDNA), which functions as an important driver of cardiac macrophage inflammation through the stimulator of interferon genes (STING) dependent mechanism. More specifically, the CVB3 infection of cardiac myocytes promotes the accumulation of extracellular mtDNA. Such myocardial mtDNA is indispensable for CVB3-infected myocytes in that it induces a macrophage inflammatory response. Mechanistically, a CVB3 infection upregulates the expression of the classical DNA sensor STING, which is predominantly localized within cardiac macrophages in VMC murine models. Myocardial mtDNA efficiently triggers STING signaling in those macrophages, resulting in strong NF-kB activation when inducing the inflammatory response. Accordingly, STING-deficient mice are able to resist CVB3-induced cardiac inflammation, exhibiting minimal inflammation with regard to their functional cardiac capacities, and they exhibit higher survival rates. Moreover, our findings pinpoint myocardial mtDNA as a central element driving the cardiac inflammation of CVB3-induced VMC, and we consider the DNA sensor, STING, to be a promising therapeutic target for protecting against RNA viral infections.

The innate immune sensor STING accelerates neointima formation via NF-κB signaling pathway.

Vascular smooth muscle cells (VSMCs) proliferation, migration, and phenotypic switching are considered crucial events in the progression of neointima formation. Stimulator of interferon genes (STING), an innate immune sensor of cyclic dinucleotides against pathogens, in neointima formation remains obscure. Here, we observed a significant increase in STING expression on the neointima of injured vessels and mouse aortic VSMCs induced by PDGF-BB. In vivo, global knockout of STING (Sting-/-) attenuated neointima formation after vascular injury. In vitro data showed that STING deficiency significantly alleviated PDGF-BB-induced proliferation and migration in VSMCs. Furthermore, these contractile marker genes were upregulated in Sting-/- VSMCs. Overexpression of STING promoted proliferation, migration, and phenotypic switching in VSMCs. Mechanistically, STING-NF-κB signaling was involved in this process. The pharmacological inhibition of STING induced by C-176 partially prevented neointima formation due to suppression of VSMCs proliferation. Taken together, STING-NF-κB axis significantly promoted proliferation, migration, and phenotypic switching of VSMCs, which may be a novel therapeutic approach to combat vascular proliferative diseases.

Oxidative stress induces MUC5AC expression through mitochondrial damage-dependent STING signaling in human bronchial epithelial cells.

Oxidative stress increases the production of the predominant mucin MUC5AC in airway epithelial cells and is implicated in the pathogenesis of bronchial asthma and chronic obstructive pulmonary disease. Oxidative stress impairs mitochondria, releasing mitochondrial DNA into the cytoplasm and inducing inflammation through the intracytoplasmic DNA sensor STING (stimulator of interferon genes). However, the role of innate immunity in mucin production remains unknown. We aimed to elucidate the role of innate immunity in mucin production in airway epithelial cells under oxidative stress. Human airway epithelial cell line (NCI-H292) and normal human bronchial epithelial cells were used to confirm MUC5AC expression levels by real-time PCR when stimulated with hydrogen peroxide (H2O2). MUC5AC transcriptional activity was increased and mitochondrial DNA was released into the cytosol by H2O2. Mitochondrial antioxidants were used to confirm the effects of mitochondrial oxidative stress where antioxidants inhibited the increase in MUC5AC transcriptional activity. Cyclic GMP-AMP synthase (cGAS) or STING knockout (KO) cells were generated to investigate their involvement. H2O2-induced MUC5AC expression was suppressed in STING KO cells, but not in cGAS KO cells. The epidermal growth factor receptor was comparably expressed in STING KO and wild-type cells. Thus, mitochondria and STING play important roles in mucin production in response to oxidative stress in airway epithelial cells.

Nile tilapia DNA sensor STING is involved in the IFN-β and AP-1 signaling pathways in the immune response dependent on DDX41

Stimulator of interferon genes (STING) plays important roles in innate immunology. In this study, we isolated the STING gene in Nile tilapia, termed OnSTING. Using quantitative RT–PCR, we explored the expression patterns of the OnSTING gene. Using dual-luciferase reporter assays, we revealed the effect of STING overexpression on nuclear factor κB (NF-κB), IFN and AP activation in HEK 293 cells. Using coimmunoprecipitation, the interaction of STING and TRIF was studied. The effect of OnSTING overexpression on the antibacterial activity in tilapia was investigated. The results showed that upon stimulation with Streptococcus agalactiae, the OnSTING transcript was upregulated in all the tested tissues. OnSTING mRNA levels were very stable from 2.5 to 8.5 dpf. Moreover, OnSTING, OnIFN and IRF3 expression was induced by LPS, Poly (I:C), S. agalactiae WC1535 and DCPS in Nile tilapia macrophages. Overexpression of OnSTING and OnDDX41 increased NF-κB activation in HEK293T cells and slightly increased IFN-β activation but had no effect on AP-1 activation. OnSTING interacted with OnDDX41 and OnTBK1. However, OnSTING did not interact with TRIF. OnSTING overexpression in vivo decreased the sensitivity of tilapia to S. agalactiae infection. These results are helpful for clarifying the innate immune response against bacterial infection in Nile tilapia.

Recent progress on the activation of the cGAS-STING pathway and its regulation by biomolecular condensation.

The cyclic guanosine monophosphate (GMP)-adenosine monophosphate (AMP) synthetase (cGAS)-stimulator of interferon genes (STING) pathway, comprising the DNA sensor cGAS, the second messenger cyclic GMP-AMP (cGAMP), and the endoplasmic reticulum (ER) adaptor protein STING, detects cytoplasmic double-stranded DNA (dsDNA) to trigger type I-interferon responses for host defense against pathogens. Previous studies defined a model for the allosteric activation of cGAS by DNA-binding, but recent work reveals other layers of mechanisms to regulate cGAS activation such as the phase condensation and metal ions, especially the discovery of Mn2+ as a cGAS activator. Activation of the 2'3'-cGAMP sensor STING requires translocating from the ER to the Golgi apparatus. The sulfated glycosaminoglycans at the Golgi are found to be the second STING ligand promoting STING oligomerization and activation in addition to 2'3'-cGAMP, while surpassed levels of 2'3'-cGAMP induce ER-located STING to form a highly organized ER membranous condensate named STING phase-separator to restrain STING activation. Here, we summarize recent advances in the regulation of cGAS-STING activation and their implications in physiological or pathological conditions, particularly focusing on the emerging complexity of the regulation.

STING is an intrinsic checkpoint inhibitor that restrains the TH17 cell pathogenic program.

SUMMARYExternal and intrinsic factors regulate the transcriptional profile of T helper 17 (TH17) cells, thereby affecting their pathogenic potential and revealing their context-dependent plasticity. The stimulator of interferon genes (STING), a component of the intracellular DNA-sensing pathway, triggers immune responses but remains largely unexplored in T cells. Here, we describe an intrinsic role of STING in limiting the TH17 cell pathogenic program. We demonstrate that non-pathogenic TH17 cells express higher levels of STING than those activated under pathogenic conditions. Activation of STING induces interleukin-10 (IL-10) production in TH17 cells, decreasing IL-17A and IL-23R expression in a type I interferon (IFN)-independent manner. Mechanistically, STING-induced IL-10 production partially requires aryl hydrocarbon receptor (AhR) signaling, while the decrease of IL-17A expression occurs due to a reduction of Rorγt transcriptional activity. Our findings reveal a regulatory function of STING in the TH17 cell activation program, proposing it as a valuable target to limit TH17-cell-mediated inflammation.

Genetic variants involved in the cGAS-STING pathway to predict outcome in patients (pts) with metastatic colorectal cancer (mCRC): Data from FIRE-3 and TRIBE trials.

3535 Background: The intracellular DNA sensor stimulator of interferon genes (STING) plays a vital role in anti-tumor immune responses by recognition of self-DNA from tumors and by-products of genomic instability. Activation of STING was reported to enhance cetuximab mediated natural killer cell activation and dendritic cell maturation. Previous reports suggested that polymorphisms of cGAS-STING can affect innate immune response. Therefore, we hypothesized that genetic variants in the cGAS-STING pathway may predict first-line treatment outcome in mCRC pts treated with bevacizumab/cetuximab-based chemotherapy. Methods: Genomic DNA from blood samples of pts enrolled in two independent randomized trials, FIRE-3 (cetuximab arm, n = 129; bevacizumab arm, n = 107) and TRIBE (bevacizumab arm, n = 215), was genotyped through the OncoArray, a customized array manufactured by Illumina including approximately 530K SNP markers. The impact on outcome of 7 selected SNPs in 3 genes involved in the STING pathway (cGAS, STING, IFNB1) was analyzed. Results: In the Cetuximab cohort, pts with STING rs1131769 any T allele (N = 29) showed significantly shorter overall survival (36.3 vs 56.07 months) compared to carriers of C/C (N = 95) in both univariate (hazard ratio [HR] = 2.08; 95% confidence interval [CI]: 1.06-4.07; p = 0.003) and multivariate (HR = 2.98; 95%CI 1.35-6.6; p = 0.00848) analysis; Pts carrying IFNB1 rs1051922 any A allele (N = 68) showed significant shorter progression-free survival (10.23 vs 14.1 months) than carriers of G/G (n = 59) in both univariate (HR = 1.87; 95%CI 1.26-2.78; p = 0.00163) and multivariate (HR = 2.03; 95%CI 1.25-3.3; p = 0.004) analysis. No association were observed in the bevacizumab cohort of TRIBE and FIRE-3. Conclusions: Our study demonstrates for the first time that STING and IFNB1 polymorphisms could predict outcomes of Cetuximab-based treatment in mCRC patients; These finding may provide insight for the combination of STING agonist and anti-EGFR treatment in mCRC patients.

Abstract PS17-17: cGAS-STING pathway protein expression in human primary breast cancer

Abstract Background: The DNA-sensing nucleotidyl transferase enzyme cyclic GMP-AMP synthase (cGAS), its second-messenger product cyclic GMP-AMP (cGAMP), and the cGAMP sensor Stimulator of interferon Genes (STING) form a major cytoplasmic DNA-sensing mechanism in human cells [Barber GN. Nat Rev Immunol, 2015]. cGAMP-activated STING oligomers bind TANK-binding kinase 1 (TBK1), translocate from the endoplasmic reticulum to perinuclear vesicles, and phosphorylate transcription factor interferon (IFN) regulatory factor 3 (IRF3), resulting in IRF3 nuclear translocation to induce expression of type I IFNs such as IFNβ. This pathway is critical for controlling innate antiviral immune responses, and is implicated in anti-tumor adaptive immunity, yet little is known regarding its function in human primary breast cancer (BC). Methods: Immunohistochemistry (IHC) staining for cGAS (ab224144), STING (CST13647), TBK1 (HPA045797), IRF3 (ab25950) and IFNβ (ab238675) was performed on tissue microarrays constructed from 18 normal human breast tissue samples and 197 primary BC samples to characterize in-situ protein expression. Positive controls were normal colon, MCF10A cells, testis tissue, MCF-7 cells and normal colon for cGAS, STING, TBK1, IRF3 and IFNβ, respectively. Substitution of primary antibodies with 0.5% normal goat serum served as negative controls. Scoring of extent of the IHC-stained area was 0 for no IHC signal, 1 for <10%, 2 for 10% to 50%, and 3 for >50% of tumor cells. IHC intensity was scored as 0 for no IHC signal, 1 for weak, 2 for moderate, and 3 for strong. A final IHC score was the product of extent score and intensity score, as described [Xia T, et al. Cell reports, 2016]. Categorical classification of high versus low final IHC scores was determined using the median as an unbiased cutoff. Associations between cGAS-STING pathway protein constituents was interrogated by Pearson’s test, and associations between STING pathway IHC expression and breast tumor clinicopathological features were by Chi-square. Results: Among 197 primary BCs, 51.5% were estrogen receptor (ER) positive/ human epidermal growth factor receptor 2 (HER2) negative, 28.4% were HER2 positive and 19.8% were triple negative BC (TNBC). Distinct cGAS-STING pathway expression patterns were observed among three BC subtypes (Table 1). Frequent high expression of cGAS-STING pathway constituents was observed in ER+/HER2- BC. By contrast, high cGAS expression was not associated with high STING expression or effector protein IFNβ in either HER2+ BC, or in TNBC. IRF3 and IFNβ expression was significantly positively associated with hormone receptor status (both P< .001); IFNβ expression was significantly inversely associated with higher grade (P=0.008) and higher mean Ki67 index (18 vs 10, P=0.005). High expression of IFNβ was positively correlated with higher levels of TBK1 and IRF3 (R=0.262 P<.001; R=0.347, P=<.001, respectively). Conclusion: To our knowledge, this is the first study to report cGAS-STING pathway in situ protein expression in primary BC, and demonstrates distinct expression patterns amongst different primary BC clinical subtypes. A limitation of this study is that IHC methods were insufficient for detecting post-translational modifications or translocation of STING pathway proteins. These results provide foundation for understanding the mechanisms of innate immune response in BC, as well as for developing new therapeutic strategies aimed at facilitating adaptive immune anti-tumor responses. Citation Format: Yu Zong, Xiaofei Liu, Glen N Barber, Mark D Pegram. cGAS-STING pathway protein expression in human primary breast cancer [abstract]. In: Proceedings of the 2020 San Antonio Breast Cancer Virtual Symposium; 2020 Dec 8-11; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2021;81(4 Suppl):Abstract nr PS17-17.

Protective Role of the Nucleic Acid Sensor STING in Pulmonary Fibrosis.

Idiopathic pulmonary fibrosis (IPF) is the most common and severe type of interstitial lung disease for which current treatments display limited efficacy. IPF is largely driven by host-derived danger signals released upon recurrent local tissue damage. Here we explored the roles of self-DNA and stimulator of interferon genes (STING), a protein belonging to an intracellular DNA sensing pathway that leads to type I and/or type III interferon (IFN) production upon activation. Using a mouse model of IPF, we report that STING deficiency leads to exacerbated pulmonary fibrosis with increased collagen deposition in the lungs and excessive remodeling factors expression. We further show that STING-mediated protection does not rely on type I IFN signaling nor on IL-17A or TGF-β modulation but is associated with dysregulated neutrophils. Together, our data support an unprecedented immunoregulatory function of STING in lung fibrosis.

Effective delivery of STING agonist using exosomes suppresses tumor growth and enhances antitumor immunity

The Stimulator of Interferon Genes (STING) pathway is implicated in the innate immune response and is important in both oncogenesis and cancer treatment. Specifically, activation of the cytosolic DNA sensor STING in antigen-presenting cells (APCs) induces a type I interferon response and cytokine production that facilitates antitumor immune therapy. However, use of STING agonists (STINGa) as a cancer therapeutic has been limited by unfavorable pharmacological properties and targeting inefficiency due to rapid clearance and limited uptake into the cytosol. Exosomes, a class of extracellular vesicles shed by all cells are under consideration for their use as effective carriers of drugs owing to their innate ability to be taken up by cells and their biocompatibility for optimal drug biodistribution. Therefore, we engineered exosomes to deliver the STING agonist cyclic GMP-AMP (iExoSTINGa), to exploit their favorable pharmacokinetics and pharmacodynamics. Selective targeting of the STING pathway in APCs with iExoSTINGa was associated with superior potency compared with STINGa alone in suppressing B16F10 tumor growth. Moreover, iExoSTINGa showed superior uptake of STINGa into dendritic cells compared with STINGa alone, which led to increased accumulation of activated CD8+ T-cells and an antitumor immune response. Our study highlights the potential of exosomes in general, and iExoSTINGa specifically, in enhancing cancer therapy outcomes.

T cell fate following Salmonella infection is determined by a STING-IRF1 signaling axis in mice

The innate immune response following infection with entero-invasive bacterial species is triggered upon release of cyclic di-guanylate monophosphate (c-di-GMP) into the host cell cytosol. Bacterial c-di-GMP activates the intracellular Sensor Stimulator of Interferon Genes (STING), encoded by Tmem173 in mice. Here we identify Interferon Regulatory Factor (IRF) 1 as a critical effector of STING-mediated microbial DNA sensing that is responsible for TH17 cell generation in the mucosal immune system. We find that STING activation induces IRF1-dependent transcriptional programs in dendritic cells (DCs) that define T cell fate determination, including induction of Gasdermin D, IL-1 family member cytokines, and enzymes for eicosanoid synthesis. Our results show that IRF1-dependent transcriptional programs in DCs are a prerequisite for antigen-specific TH17 subspecification in response to microbial c-di-GMP and Salmonella typhimurium infection. Our identification of a STING-IRF1 signaling axis for adaptive host defense control will aid further understanding of infectious disease mechanisms.

Pro-inflammation Associated with a Gain-of-Function Mutation (R284S) in the Innate Immune Sensor STING

The cellular sensor stimulator of interferon genes (STING) initiates type I interferon (IFN) and cytokine production following association with cyclic dinucleotides (CDNs) generated from intracellular bacteria or via a cellular synthase, cGAS, after binding microbial or self-DNA. Although essential for protecting the host against infection, unscheduled STING signaling is now known to be responsible for a variety of autoinflammatory disorders. Here, we report a gain-of-function mutation in STING (R284S), isolated from a patient who did not require CDNs to augment activity and who manifested a constitutively active phenotype. Control of the Unc-51-like autophagy activating kinase 1 (ULK1) pathway, which has previously been shown to influence STING function, was potently able to suppress STING (R284S) activity to alleviatecytokine production. Our findings add to the growinglist of inflammatory syndromes associated with spontaneous STING signaling and provide a therapeutic strategy for the treatment of STING-induced inflammatory disease.

Development of a Validated Interferon Score Using NanoString Technology.

Chronic elevation of interferon (IFN)-response genes (IRG) in a subset of patients with systemic immune-dysregulatory diseases, including the Mendelian Type-I IFN-mediated autoinflammatory diseases and some autoimmune diseases suggest a causative role of excessive IFN signaling in the disease pathogenesis and as target for treatment. We developed a 28-IFN response gene scoring system to calculate either a standardized or geomean score by customizing a NanoString assay to quantify the expression of putative IRGs. The gene targets were selected in patients with the IFN-mediated disease chronic atypical neutrophilic dermatosis with lipodystrophy and elevated temperature (CANDLE) and an adult patient with chronic hepatitis C who received the first dose of pegylated interferon alpha-2a. The putative target genes were validated in patients with STING-associated vasculopathy with onset in infancy (SAVI), a monogenic autoinflammatory disease caused by gain-of-function mutations in TMEM173 that encodes the viral sensor stimulator of IFN genes (STING), and had low expression in clinically active patients with the monogenic IL-1-mediated autoinflammatory disease, neonatal-onset multisystem inflammatory disease (NOMID) and in healthy controls. The score calculation on the NanoString assay is rapid and showed high reproducibility and low intra-, and interassay variability. The utility of this 28-gene IFN score may be explored in the diagnosis of patients with presumed interferonopathies and as a biomarker to assess disease activity, long-term outcome, and treatment responses.

STING activation enhances cetuximab-mediated NK cell activation and DC maturation and correlates with HPV+ status in head and neck cancer

ObjectivesThe intracellular DNA sensor stimulator of interferon genes (STING) has recently been shown to play a vital role in anti-viral and anti-tumor immune responses stimulating cytokine production. While human papillomavirus (HPV) is a causative agent for a subset of head and neck squamous cell carcinoma (HNSCC) with unique etiology and clinical outcome, how the STING pathway is regulated in a virus-induced tumor microenvironment is not well understood. Since STING inactivation likely reflects immunoescape via innate immunity, we hypothesized that its restoration would improve efficacy of the immune modulatory monoclonal antibody (mAb), cetuximab. Materials and methodsWe correlated STING protein expression with clinical parameters by immunohistochemistry (n = 106) and its mRNA expression from The Cancer Genome Atlas (TCGA) in HNSCC tissue specimens. STING protein expression was tested for association with cancer-specific survival (CSS). We further examined the impact of STING activation on cetuximab-mediated immunity using an in vitro NK:DC:tumor cells co-culture system. ResultsIn this study, we found that expression of STING both at the protein and mRNA level was higher in HPV positive (HPV+) specimens but unrelated to TNM stage or cancer-specific survival. Our in vitro studies verified that STING activation enhanced cetuximab mediated NK cell activation and DC maturation. ConclusionOur findings suggest a novel role of STING in HPV-related carcinogenesis, in which activation of the STING signaling pathway may facilitate anti-tumor response in HNSCC patients, particularly in combination with therapeutic monoclonal antibodies (mAbs) such as cetuximab, an epidermal growth factor receptor (EGFR) inhibitor.

The cytosolic sensor STING is required for intestinal homeostasis and control of inflammation.

STING (stimulator of interferon genes) is a cytosolic sensor for cyclic dinucleotides and also an adaptor molecule for intracellular DNA receptors. Although STING has important functions in the host defense against pathogens and in autoimmune diseases, its physiological relevance in intestinal homeostasis is largely unknown. In this study, we show that STING-/- mice presented defective protective mechanisms of intestinal mucosa, including decreased number of goblet cells, diminished mucus production, and lower levels of secretory IgA, when compared with wild-type (WT) mice. Fecal content and microbiota DNA could activate STING, indicating a role of this molecule in gut. Microbiota composition was altered in STING-/- mice toward a more inflammatory profile, evidencing a reduction in the Allobacolum and Bifidobacterium groups along with increase in Disulfovibrio bacteria. Absence of STING lead to decrease in induced intraepithelial lymphocytes (IEL) and to increase in group 1 innate lymphoid cell (ILC1) as well as ILC3 frequencies and decrease in ILC2 in the colon. Development and function of Foxp3+ and LAP+ regulatory T cells were also compromised in STING-/- mice. Moreover, these mice were highly susceptible to dextran sodium sulfate-induced colitis, T-cell-induced colitis, and enteric Salmonella typhimurium infection when compared with WT animals. Therefore, our results identify an important role of STING in maintaining gut homeostasis and also a protective effect in controlling gut inflammation.

B Cells Producing Type I IFN Modulate Macrophage Polarization in Tuberculosis.

In addition to their well-known function as antibody-producing cells, B lymphocytes can markedly influence the course of infectious or noninfectious diseases via antibody-independent mechanisms. In tuberculosis (TB), B cells accumulate in lungs, yet their functional contribution to the host response remains poorly understood. To document the role of B cells in TB in an unbiased manner. We generated the transcriptome of B cells isolated from Mycobacterium tuberculosis (Mtb)-infected mice and validated the identified key pathways using in vitro and in vivo assays. The obtained data were substantiated using B cells from pleural effusion of patients with TB. B cells isolated from Mtb-infected mice displayed a STAT1 (signal transducer and activator of transcription 1)-centered signature, suggesting a role for IFNs in B-cell response to infection. B cells stimulated in vitro with Mtb produced type I IFN, via a mechanism involving the innate sensor STING (stimulator of interferon genes), and antagonized by MyD88 (myeloid differentiation primary response 88) signaling. In vivo, B cells expressed type I IFN in the lungs of Mtb-infected mice and, of clinical relevance, in pleural fluid from patients with TB. Type I IFN expression by B cells induced an altered polarization of macrophages toward a regulatory/antiinflammatory profile in vitro. In vivo, increased provision of type I IFN by B cells in a murine model of B cell-restricted Myd88 deficiency correlated with an enhanced accumulation of regulatory/antiinflammatory macrophages in Mtb-infected lungs. Type I IFN produced by Mtb-stimulated B cells favors macrophage polarization toward a regulatory/antiinflammatory phenotype during Mtb infection.

STING Senses Microbial Viability to Orchestrate Stress-Mediated Autophagy of the Endoplasmic Reticulum

Constitutive cell-autonomous immunity in metazoans predates interferon-inducible immunity and comprises primordial innate defense. Phagocytes mobilize interferon-inducible responses upon engagement of well-characterized signaling pathways by pathogen-associated molecular patterns (PAMPs). The signals controlling deployment of constitutive cell-autonomous responses during infection have remained elusive. Vita-PAMPs denote microbial viability, signaling the danger of cellular exploitation by intracellular pathogens. We show that cyclic-di-adenosine monophosphate in live Gram-positive bacteria is a vita-PAMP, engaging the innate sensor stimulator of interferon genes (STING) to mediate endoplasmic reticulum (ER) stress. Subsequent inactivation of themechanistic target of rapamycin mobilizes autophagy, which sequesters stressed ER membranes, resolves ER stress, and curtails phagocyte death. This vita-PAMP-induced ER-phagy additionally orchestrates an interferon response by localizing ER-resident STING to autophagosomes. Our findings identify stress-mediated ER-phagy as a cell-autonomous response mobilized by STING-dependent sensing of a specific vita-PAMP and elucidate how innate receptors engage multilayered homeostatic mechanisms topromote immunity and survival after infection.

Endosomal RNA-sensing toll-like receptor stimulated T cells play a unique role in autoimmune manifestations in murine model of DNA accrual.

Abstract DNaseII is a lysosomal DNase that plays a critical role in immune homeostasis. DNaseII deficiency in mice causes embryonic lethality, due to severe anemia associated with excessive production of type I interferon (IFN), and can be rescued by IFN receptor knockout (IFNaR). The DNaseII/IFNaR double knockout (DKO) mice develop a late-onset inflammatory arthritis, dependent on the cytosolic DNA sensor STING (Stimulator of Interferon Genes). They also develop early-onset clinical manifestations of systemic autoimmunity including disrupted hematopoiesis and B cell lymphopoiesis, splenomegaly (due to increased accumulation of Ter119+ cells), and the production of anti-nuclear antibodies (ANA), through a mechanism that is STING-independent, Unc93B1-dependent and likely to result from endosomal RNA-sensing TLRs. Bone marrow (BM) chimera studies have shown that the DNaseII deficiency is essential in both radioresistant host cells and hematopoietic donor cells for all disease manifestations. Development of arthritis requires the presence of STING in both host and donor. The TLR-dependent defects require TLR expression in only the hematopoietic compartment but still need DNaseII−/− recipients. Remarkably transplantation of DKO spleen fragments or injection of DKO spleen cells alone into DNaseII-sufficient Rag−/− mice leads to splenomegaly and disrupted hematopoiesis and lymphopoiesis. Moreover DKO T cells are sufficient to transfer these TLR-associated defects to the Rag−/− mice. As activated T cells are known to be radioresistant, T cells activated in a DKO environment may account for the radioresistant host component required to precipitate the TLR-dependent disease manifestations.

Impaired STING Pathway in Human Osteosarcoma U2OS Cells Contributes to the Growth of ICP0-Null Mutant Herpes Simplex Virus

Human herpes simplex virus 1 (HSV-1) is a widespread pathogen, with 80% of the population being latently infected. To successfully evade the host, the virus has evolved strategies to counteract antiviral responses, including the gene-silencing and innate immunity machineries. The immediately early protein of the virus, infected cell protein 0 (ICP0), plays a central role in these processes. ICP0 blocks innate immunity, and one mechanism is by degrading hostile factors with its intrinsic E3 ligase activity. ICP0 also functions as a promiscuous transactivator, and it blocks repressor complexes to enable viral gene transcription. For these reasons, the growth of a ΔICP0 virus is impaired in most cells, except cells of the human osteosarcoma cell line U2OS, and it is only partially impaired in cells of the human osteosarcoma cell line Saos-2. We found that the two human osteosarcoma cell lines that supported the growth of the ΔICP0 virus failed to activate innate immune responses upon treatment with 2'3'-cyclic GAMP (2'3'-cGAMP), the natural agonist of STING (i.e., stimulator of interferon genes) or after infection with the ΔICP0 mutant virus. Innate immune responses were restored in these cells by transient expression of the STING protein but not after overexpression of interferon-inducible protein 16 (IFI16). Restoration of STING expression resulted in suppression of ΔICP0 virus gene expression and a decrease in viral yields. Overexpression of IFI16 also suppressed ΔICP0 virus gene expression, albeit to a lesser extent than STING. These data suggest that the susceptibility of U2OS and Saos-2 cells to the ΔICP0 HSV-1 is in part due to an impaired STING pathway.IMPORTANCE The DNA sensor STING plays pivotal role in controlling HSV-1 infection both in cell culture and in mice. The HSV-1 genome encodes numerous proteins that are dedicated to combat host antiviral responses. The immediate early protein of the virus ICP0 plays major role in this process as it targets hostile host proteins for degradation with its E3 ligase activity, and it disrupts repressor complexes via protein-protein interaction to enable viral gene transcription. Therefore, the ΔICP0 HSV-1 virus is defective for growth in most cells, except the human osteosarcoma cell lines U2OS and Saos-2. We found that both cell lines that support ΔICP0 virus infection have defects in the STING DNA-sensing pathway, which partially accounts for the rescue of the ΔICP0 virus growth. Restoration of STING expression in these cells rescued innate immunity and suppressed ΔICP0 virus infection. This study underscores the importance of STING in the control of HSV-1.

Hepatitis B virus polymerase disrupts K63-linked ubiquitination of STING to block innate cytosolic DNA-sensing pathways.

The cellular innate immune system recognizing pathogen infection is essential for host defense against viruses. In parallel, viruses have developed a variety of strategies to evade the innate immunity. The hepatitis B virus (HBV), a DNA virus that causes chronic hepatitis, has been shown to inhibit RNA helicase RIG-I-mediated interferon (IFN) induction. However, it is still unknown whether HBV could affect the host DNA-sensing pathways. Here we report that in transiently HBV-transfected Huh7 cells, the stably HBV-producing cell line HepAD38, and HBV-infected HepaRG cells and primary human hepatocytes, HBV markedly interfered with IFN-β induction and antiviral immunity mediated by the stimulator of interferon genes (STING), which has been identified as a central factor in foreign DNA recognition and antiviral innate immunity. Screening analysis demonstrated that the viral polymerase (Pol), but not other HBV-encoded proteins, was able to inhibit STING-stimulated interferon regulatory factor 3 (IRF3) activation and IFN-β induction. Moreover, the reverse transcriptase (RT) and the RNase H (RH) domains of Pol were identified to be responsible for the inhibitory effects. Furthermore, Pol was shown to physically associate with STING and dramatically decrease the K63-linked polyubiquitination of STING via its RT domain without altering the expression level of STING. Taken together, these observations suggest that besides its inherent catalytic function, Pol has a role in suppression of IFN-β production by direct interaction with STING and subsequent disruption of its K63-linked ubiquitination, providing a new mechanism for HBV to counteract the innate DNA-sensing pathways. Although whether and how HBV infection induces the innate immune responses are still controversial, it has become increasingly clear that HBV has developed strategies to counteract the pattern recognition receptor-mediated signaling pathways. Previous studies have shown that type I IFN induction activated by the host RNA sensors could be inhibited by HBV. However, it remains unknown whether HBV as a DNA virus utilizes evasion mechanisms against foreign DNA-elicited antiviral signaling. In recent years, the cytosolic DNA sensor and key adaptor STING has been demonstrated to be essential in multiple foreign DNA-elicited innate immune signalings. Here, for the first time, we report STING as a new target of HBV to antagonize IFN induction and identify the viral polymerase responsible for the inhibitory effect, thus providing an additional molecular mechanism by which HBV evades the innate immunity; this implies that in addition to its inherent catalytic function, HBV polymerase is a multifunctional immunomodulatory protein.