Interferon-stimulated Genes Research Articles

STING orchestrates microglia polarization via interaction with LC3 in autophagy after ischemia

AbstractAutophagy has both protective and pathogenetic effects on injury caused by cerebral ischemia/reperfusion (I/R). Our previous research has indicated that stimulator of interferon genes (STING) could orchestrate microglia polarization following middle cerebral artery occlusion. However, it remains largely unexplored whether STING balances microglial polarization by regulating autophagy in brain I/R injury. Here, STING was observed to show an up-regulation in the microglia from mice subjected to experimental ischemic stroke. Strikingly, the deletion of STING led to the significant skewness of microglia activated by ischemia from a pro- to anti-inflammatory state and substantially alleviated ischemia-induced infarction and neuronal injury. In addition, STING-null mice can restore long-term neurobehavioral function. Then, the crosstalk between neuroinflammation and microglia autophagy was analyzed. The differential activity of autophagy in wild-type and STING-knockout (KO) mice or primary microglia was largely reversed when STING was restored in microglia. Irritating autophagy by rapamycin skewed the anti‑inflammatory state induced by STING-KO to a pro‑inflammatory state in microglia. Furthermore, microtubule-associated protein light-chain-3 (LC3) was identified as the key factor in the STING regulation of autophagy by glutathione-S-transferase (GST) pull-down analysis. Mechanically, STING can directly interact with LC3 through the STING transmembrane domain (1-139aa). Herein, current data determine the pivotal role of autophagy, specifically via LC3 protein, in the regulation of microglial phenotypic transformation by STING. These findings may provide a possible treatment target for delaying the progression of ischemic stroke.

The Antiviral Effects of Heat-Killed Lactococcus lactis Strain Plasma Against Dengue, Chikungunya, and Zika Viruses in Humans by Upregulating the IFN-α Signaling Pathway

The growing risk of contracting viral infections due to high-density populations and ecological disruptions, such as climate change and increased population mobility, has highlighted the necessity for effective antiviral treatment and preventive measures against Dengue virus (DENV), Chikungunya virus (CHIKV), and Zika virus (ZIKV). Recently, there has been increasing attention on the use of probiotics as a potential antiviral option to reduce virus infections. The present study aimed to assess the immunomodulatory effects of heat-killed Lactococcus lactis strain plasma (LC-Plasma) on peripheral blood mononuclear cells (PBMCs) and its subsequent antiviral response against DENV, CHIKV, and ZIKV. To evaluate the immunomodulatory effects of LC-Plasma on PBMCs isolated from healthy individuals, PBMCs were cultured at a density of 2 × 105 cells/well and stimulated with 10 µg/mL of LC-Plasma. LC-plasma-stimulated PBMCs demonstrated elevated interferon-alpha (IFN-α) production and cluster of differentiation 86 (CD86) and human leukocyte antigen-DR isotype (HLA-DR) upregulation, potentially linked to plasmacytoid dendritic cell (pDC) activation. The replication of DENV, CHIKV, and ZIKV was dose-dependently inhibited when Huh-7 cells were stimulated with LC-Plasma-stimulated PBMC supernatant (LCP Sup). IFN-stimulated gene (ISG) expression, including IFN-stimulated gene 15 (ISG15), IFN-stimulated exonuclease gene 20 (ISG20), IFN-induced transmembrane protein 1 (IFITM-1), myxovirus resistance protein A (MxA), and radical S-adenosyl methionine domain-containing protein 2 (RSAD2), was significantly upregulated in LCP Sup-stimulated Huh-7 cells. Findings from this study indicate that LC-Plasma has the potential to induce IFN-α production, leading to an enhancement in the expression of ISGs and contributing to a broad-spectrum antiviral response. Thus, LC-Plasma may serve as a rational adjunctive treatment to ameliorate viral diseases, warranting future clinical trials.

A Phosphotriester‐Masked Dideoxy‐cGAMP Derivative as a Cell‐Permeable STING Agonist

2’3’‐cyclic GMP‐AMP (cGAMP) is a cyclic dinucleotide second messenger in which guanosine and adenosine are connected by one 3’‐5’ and one 2’‐5’ phosphodiester linkage. It is formed in the cytosol upon detection of pathogenic DNA by the enzyme guanosine‐monophosphate‐adenosine monophosphate synthase (cGAS). cGAMP subsequently binds to the adaptor protein stimulator of interferon genes (STING) to elicit an innate immune response leading to the production of type I interferons and cytokines. STING agonists are a highly promising avenue for an immuno‐oncological anticancer therapy. A particular challenge with cyclic dinucleotide STING agonists are the two negative charges of the phosphodiester linkages, which strongly reduce the ability of such compounds to penetrate cell membranes. The development of cell‐permeable STING agonists that can stimulate the immune system, enhancing their anticancer potency is currently of utmost importance in the field. Here, we report the development of a dideoxy derivative of cGAMP as a phosphotriester prodrug, where the negative charge of the phosphate backbone has been masked with a thioester. We found that this thioester‐protected compound features a dramatic increase in its cellular potency that rises from EC50 = 5 mM to 25 nM. The new compound is envisioned to enable an efficient STING‐agonist‐based anticancer therapy.

A Phosphotriester‐Masked Dideoxy‐cGAMP Derivative as a Cell‐Permeable STING Agonist

2’3’‐cyclic GMP‐AMP (cGAMP) is a cyclic dinucleotide second messenger in which guanosine and adenosine are connected by one 3’‐5’ and one 2’‐5’ phosphodiester linkage. It is formed in the cytosol upon detection of pathogenic DNA by the enzyme guanosine‐monophosphate‐adenosine monophosphate synthase (cGAS). cGAMP subsequently binds to the adaptor protein stimulator of interferon genes (STING) to elicit an innate immune response leading to the production of type I interferons and cytokines. STING agonists are a highly promising avenue for an immuno‐oncological anticancer therapy. A particular challenge with cyclic dinucleotide STING agonists are the two negative charges of the phosphodiester linkages, which strongly reduce the ability of such compounds to penetrate cell membranes. The development of cell‐permeable STING agonists that can stimulate the immune system, enhancing their anticancer potency is currently of utmost importance in the field. Here, we report the development of a dideoxy derivative of cGAMP as a phosphotriester prodrug, where the negative charge of the phosphate backbone has been masked with a thioester. We found that this thioester‐protected compound features a dramatic increase in its cellular potency that rises from EC50 = 5 mM to 25 nM. The new compound is envisioned to enable an efficient STING‐agonist‐based anticancer therapy.

Abstract 4137846: Smooth muscle expression of RNA editing enzyme ADAR1 controls vascular integrity and progression of atherosclerosis

Mapping the genomic architecture of complex disease has been predicated on the understanding that genetic variants influence disease risk through modifying gene expression. However, recent discoveries have revealed that a significant burden of disease heritability in common autoinflammatory disorders and coronary artery disease is mediated through genetic variation modifying post-transcriptional modification of RNA through adenosine-to-inosine (A-to-I) RNA editing. This common RNA modification is catalyzed by ADAR enzymes, where ADAR1 edits specific immunogenic double stranded RNA (dsRNA) to prevent activation of the double strand RNA (dsRNA) sensor MDA5 ( IFIH1 ) and stimulation of an interferon stimulated gene (ISG) response. Multiple lines of human genetic data indicate impaired RNA editing and increased dsRNA sensing to be an important mechanism of coronary artery disease (CAD) risk. Here, we provide a crucial link between observations in human genetics and mechanistic cell biology leading to progression of CAD. Through analysis of human atherosclerotic plaque, we implicate the vascular smooth muscle cell (SMC) to have a unique requirement for RNA editing, and that ISG induction occurs in SMC phenotypic modulation, implicating MDA5 activation. Through culture of human coronary artery SMCs, generation of a conditional SMC specific Adar1 deletion mouse model on a pro-atherosclerosis background, and with incorporation of single cell RNA sequencing cellular profiling, we further show that Adar1 controls SMC phenotypic state, is required to maintain vascular integrity, and controls progression of atherosclerosis and vascular calcification. Our data implicates MDA5 activation to occur in SMC phenotypic modulation and accelerates formation of chondromyocytes in a distinct cellular lineage trajectory — indicating a cell type and context specific requirement of RNA editing. Through this work, we describe a fundamental new mechanism of CAD, where RNA editing and sensing of dsRNA in a cell type and context specific mechanism mediates disease progression, bridging our understanding of human genetics and disease causality.

Type I Interferon Drives a Cellular State Inert to TCR‐Stimulation and Could Impede Effective T‐Cell Differentiation in Cancer

ABSTRACTBackgroundHead and neck squamous cell carcinoma (HNSCC) is linked to human papillomavirus (HPV) infection. HPV‐positive and HPV‐negative HNSCC exhibit distinct molecular and clinical characteristics. Although checkpoint inhibitors have shown efficiency in recurrent/metastatic HNSCC, response variability persists regardless of HPV status. This study aimed to explore the CD8+ T‐cell landscape in HPV‐negative HNSCC.MethodsWe performed simultaneous single‐cell RNA and TCR sequencing of CD8+ tumor‐infiltrating lymphocytes (TILs) from treatment‐naïve HPV‐negative HNSCC patients. Additionally, cells were stimulated ex vivo, which allowed for the tracking of clonal transcriptomic responses.ResultsOur analysis identified a subset of CD8+ TILs highly enriched for interferon‐stimulated genes (ISG). TCR analysis revealed ISG cells are clonally related to a population of granzyme K (GZMK)‐expressing cells. However, unlike GZMK cells, which exhibited rapid effector‐like phenotypes following stimulation, ISG cells were transcriptionally inert. Additionally, ISG cells showed specific enrichment within tumor and were found across multiple tumor entities.ConclusionsISG‐enriched CD8+ TILs are a consistent feature of various tumor entities. These cells are poorly understood but possess characteristics that may impact antitumor immunity. Understanding the unique properties and functionality of ISG cells could offer innovative treatment approaches to improve patient outcomes in HPV‐negative HNSCC and other cancer types.

TMOD-32. MURINE MODELLING OF THE STING PATHWAY IN GLIOMAS

Abstract The stimulator of interferon genes (STING) pathway is a critical component of innate antitumor immunity through the recognition of pathogenic cytosolic DNA and stimulating the production of type 1 interferons and pro-inflammatory cytokines. We have previously shown that STING signaling is silenced in the neoplastic compartment of the GBM tumor microenvironment, but remains intact in vascular and immune cells. In human patient derived cell lines, such silencing appears to be mediated by hypermethylation of a region of the STING promoter and can be reversed by treatment with both the DNA methyltransferase (DNMT) inhibitor Decitabine and DNMT1-selective inhibitor GSK-3685032. These results suggest that neoplastic cell STING promoter methylation represents a therapeutic vulnerability that can be exploited by epigenetic therapies to inflame the glioma tumor microenvironment. Murine models are critical tools for exploring the translational implications of STING silencing in vivo. Distinct mouse models have been developed to optimally represent specific aspects of human glioma biology. Here we characterize the STING pathway in commonly used syngeneic murine glioma models, including CT2A, GL261, SB28, and SMA560. We show that the expression of STING pathway components varies significantly across these lines. Importantly, unlike in humans, murine STING (mSTING) expression is not suppressed by promoter methylation and does not respond to decitabine treatment. Additionally, the mSTING protein is expressed in neoplastic cells in vivo. These results demonstrate a fundamental difference in epigenetic regulation of STING between human and murine glioma cells, and suggest that murine modeling of STING epigenetics requires the use of patient derived cell lines implanted in immunodeficient mice. Meanwhile, immune competent syngeneic models are appropriate for exploring the tumor immune microenvironment but would require non-epigenetic approaches (e.g. genetic knock-outs/knock-ins) for modulating STING expression. Humanized mice represent a promising emerging approach for faithfully recapitulating both epigenetic control and immune environment of human gliomas.

IMMU-19. PRECLINICAL THERAPEUTIC ACTIVITY AND PET IMAGING OF STING AGONIST 8803 IN GLIOBLASTOMA

Abstract STING (stimulator of interferon genes) activation triggers interferon (IFN) release within the tumor microenvironment from myeloid cells and other cell types, inducing proinflammatory anti-tumor immune responses. The small molecule STING agonist, 8803, elicits long-term survival in 56% (QPP4; p=0.0003) to 100% (QPP8; p<0.0001) of mice with orthotopically implanted immune checkpoint-resistant glioblastoma models after 2-3 doses. In humanized mice, in which glioblastoma-cell STING is epigenetically silenced, 8803 therapeutic activity is maintained. The combination therapy of 8803 with anti-PD-1 relatively further enhances survival in immune checkpoint-resistant models. Ex vivo flow cytometry and proteomic sequential multiplex profiling during the therapeutic window demonstrate global immunological reprogramming, specific tumor-antigen immune responses, increased tumor immune effector trafficking, and release of IFN. It has been shown that IFN signaling augments intracellular nucleotide metabolism and increases [18F]-FLT uptake in pancreatic cancer. We evaluated the longitudinal [18F]-FLT uptake in CT-2A-bearing mice using MRI/PET/CT at days 0, 2, and 4 after intratumoral 8803 delivery. Brain images were acquired before and after [18F]-FLT tracer and 8803 administrations. The CT was used for PET attenuation/correction, and the MRI was used for tumor segmentation. Regional analysis of PET-derived [18F]-FLT SUV maps used advanced image processing approaches as a function of time to enhance the voxel-by-voxel interpretation of the acquired images. [18F]-FLT uptake increased from baseline in all mice treated with 8803 compared to control mice with a peak at 48-96 hours. Concordant sequential multiplex immunofluorescence demonstrated that the STING expression was prominent along the tumor vasculature, myeloid, and tumor cells. p-IRF-3 expression, reflective of STING pathway activation, showed a diffuse pattern after treatment. Based on the preclinical data, PET imaging at 48-96 hours post-treatment represents the appropriate imaging modality and timepoint to maximize the ability to detect STING-dependent changes for a therapeutic strategy that reprograms the glioblastoma microenvironment and improves survival across multiple preclinical models of glioblastoma.

IMMU-54. THERAPEUTIC TARGETING OF STING IN MENINGIOMAS

Abstract Up to one-quarter of meningiomas recur within five years of resection; however, there are currently no established adjuvant therapies for lesions that are intractable to surgery and radiation. To identify novel treatment approaches, scRNA-sequencing datasets were reanalyzed from thirteen meningiomas and nine adjacent dura samples including >30,000 immune cells from varying WHO grades and molecular subtypes. In contrast to gliomas, immune-inducing STING (stimulator of interferon genes) was expressed within the meningioma tumor cells, consistent with DNA methylation profiling that showed hypomethylation of the STING promotor in 93% of cases (267 of 288 meningiomas; beta < 0.2). Meningiomas in the immune-enriched subgroup additionally exhibited significant hypomethylation in the 3’ UTR of this gene (p = 1.9 x 10-9). Using sequential multiplex immune fluorescence, STING expression was confirmed in neoplastic, myeloid, and endothelial cells within all grades of meningiomas, but not in adjacent dura and brain. Downstream STING pathway activation markers such as p-IRF3 were partially expressed, suggesting that further activation of the STING pathway may offer therapeutic benefits. Ex vivo single-cell suspensions of resected human meningiomas treated with the STING agonist 8803 induced 25-45% growth inhibition in 7/8 cases within 72 hours (p < 0.001; two-way ANOVA). There was no growth inhibition of the meningioma cell lines IOMM-LEE and CH157 treated with 8803, which lack immune cells and contain only clonal neoplastic cells. However, when the IOMM-LEE meningioma cells were implanted in vivo, a single dose of 8803 induced a >50% reduction in tumor volume relative to the vehicle control injection at multiple time points (p < 0.001; two-way ANOVA) indicating that the immune system is an essential component of STING efficacy in these tumors. Our results reveal widespread expression of STING in neoplastic and myeloid populations in meningiomas and clarify a new treatment indication for use of the STING agonist 8803.

Tandem‐Controlled Dynamic DNA Assembly Enables Temporally‐Selective Orthogonal Regulation of cGAS–STING Stimulation

Despite advances in the controlled reconfiguration of DNA structures for biological applications, the dearth of strategies that allow for orthogonal regulation of immune pathways remains a challenge. Here, we report for the first time an endogenous and exogenous tandem‐regulated DNA assembly strategy that enables orthogonally controlled stimulation of the cyclic GMP‐AMP synthase (cGAS)–stimulator of interferon genes (STING) pathway. A DNA motif containing two palindromic sequences is engineered with an abasic site (AP)‐connected blocking sequence to inhibit its self‐assembly function, while apurinic/apyrimidinic endonuclease 1 (APE1)‐triggered enzymatic cleavage of the AP site enables the reconfiguration and self‐assembly of DNA motif into long double‐stranded structures, thus realizing allosteric activation of the catalytic activity of cGAS to produce 2’3’‐cyclic‐GMP‐AMP for STING stimulation. Importantly, we demonstrate that APE1‐regulated DNA assembly allows for cell‐selective activation of cGAS–STING signaling. Furthermore, by re‐engineering the DNA motif with a photocleavable group, enzyme‐triggered DNA assembly allows the cGAS–STING stimulation to operate (switched "ON''), whereas light‐mediated fragmentation of the double‐stranded DNA enables termination of such stimulation (switched "OFF"), thereby achieving orthogonal control over immune regulation. This work highlights an endogenous and exogenous tandem regulated strategy to modulate the cGAS–STING pathway in an orthogonally controlled manner.

Tandem‐Controlled Dynamic DNA Assembly Enables Temporally‐Selective Orthogonal Regulation of cGAS–STING Stimulation

Despite advances in the controlled reconfiguration of DNA structures for biological applications, the dearth of strategies that allow for orthogonal regulation of immune pathways remains a challenge. Here, we report for the first time an endogenous and exogenous tandem‐regulated DNA assembly strategy that enables orthogonally controlled stimulation of the cyclic GMP‐AMP synthase (cGAS)–stimulator of interferon genes (STING) pathway. A DNA motif containing two palindromic sequences is engineered with an abasic site (AP)‐connected blocking sequence to inhibit its self‐assembly function, while apurinic/apyrimidinic endonuclease 1 (APE1)‐triggered enzymatic cleavage of the AP site enables the reconfiguration and self‐assembly of DNA motif into long double‐stranded structures, thus realizing allosteric activation of the catalytic activity of cGAS to produce 2’3’‐cyclic‐GMP‐AMP for STING stimulation. Importantly, we demonstrate that APE1‐regulated DNA assembly allows for cell‐selective activation of cGAS–STING signaling. Furthermore, by re‐engineering the DNA motif with a photocleavable group, enzyme‐triggered DNA assembly allows the cGAS–STING stimulation to operate (switched "ON''), whereas light‐mediated fragmentation of the double‐stranded DNA enables termination of such stimulation (switched "OFF"), thereby achieving orthogonal control over immune regulation. This work highlights an endogenous and exogenous tandem regulated strategy to modulate the cGAS–STING pathway in an orthogonally controlled manner.

Bifunctional Metal–Organic Framework Synergistically Enhances Radiotherapy and Activates STING for Potent Cancer Radio‐Immunotherapy

AbstractThe activation of the stimulator of interferon genes (STING) protein by cyclic dinucleotide metabolites plays a critical role in antitumor immunity. However, synthetic STING agonists like 4‐(5,6‐dimethoxybenzo[b]thiophen‐2‐yl)‐4‐oxobutanoic acid (MSA‐2) exhibit suboptimal pharmacokinetics and fail to sustain STING activation in tumors for effective antitumor responses. Here, we report the design of MOF/MSA‐2, a bifunctional MSA‐2 conjugated nanoscale metal–organic framework (MOF) based on Hf6 secondary building units (SBUs) and hexakis(4′‐carboxy[1,1′‐biphenyl]‐4‐yl)benzene bridging ligands, for potent cancer radio‐immunotherapy. By leveraging the high‐Z properties of the Hf6 SBUs, the MOF enhances the therapeutic effect of X‐ray radiation and elicits potent immune stimulation in the tumor microenvironment. MOF/MSA‐2 further enhances radiotherapeutic effects of X‐rays by enabling sustained STING activation and promoting the infiltration and activation of immune cells in the tumors. MOF/MSA‐2 plus low‐dose X‐ray irradiation elicits strong STING activation and potent tumor regression, and when combined with an immune checkpoint inhibitor, effectively suppresses both primary and distant tumors through systemic immune activation.

Common Cold Coronavirus 229E Induces Higher Interferon Stimulating Gene Responses in Human Nasal Epithelial Cells from Patients with Chronic Rhinosinusitis with Polyposis.

Viral infections have long been implicated in the development of chronic rhinosinusitis with nasal polyps (CRSwNP). Given widespread exposure to the common cold coronavirus 229E (HCoV229E), we sought to investigate how HCoV-229E is cleared and stimulates interferon pathways in air-liquid interface (ALI) cultures from patients with CRSwNP. The objective of this study was to identify whether viral clearance and ISG expression is different in ALI cultures from donors with CRSwNP compared with controls. Plaque assays were used to quantify infectious virus released by infected air-liquid interface (ALI) cultures derived from patients with CRSwNP compared to patients without CRS (controls). Additionally, mock and induced levels of Interferon Stimulated Genes (ISGs) mRNA following HCoV-229E infection were quantified by RT-qPCR. Quantification of infectious virus by plaque assay reveals that CRSwNP ALI cultures were equally susceptible to HCoV-229E infection, and surprisingly viral titers dropped significantly faster than in the control ALI cultures. We further demonstrate that this accelerated viral clearance correlates with increased mRNA expression of at least 4 ISGs following viral infection in the CRSwNP ALIs compared to the control ALIs. This study paradoxically demonstrates that ALI cultures from patients with CRSwNP are more efficient at clearing the common cold HCoV-229E virus compared to controls. We also demonstrate significantly increased ISG mRNA expression following HCoV-229E infection in CRSwNP. These findings call for further investigation into the effect of unimpaired interferon signaling on the type 2 inflammatory environment in patients with CRSwNP.

UBR7 E3 Ligase Suppresses Interferon-β Mediated Immune Signaling by Targeting Sp110 in Hepatitis B Virus-Induced Hepatocellular Carcinoma.

A newly discovered E3 ubiquitin ligase, UBR7, plays a crucial role in histone H2BK120 monoubiquitination. Here, we report a novel function of UBR7 in promoting hepatitis B virus (HBV) pathogenesis, which further leads to HBV-induced hepatocellular carcinoma (HCC). Transcriptomics analysis from HCC patients revealed the deregulation of UBR7 in cancer. Remarkably, targeting UBR7, particularly its catalytic function, led to a significant decrease in viral copy numbers. We also identified the speckled family protein Sp110 as an important substrate of UBR7. Notably, Sp110 has been previously shown to be a resident of promyelocytic leukemia nuclear bodies (PML-NBs), where it remains SUMOylated, and during HBV infection, it undergoes deSUMOylation and exits the PML body. We observed that UBR7 ubiquitinates Sp110 at critical residues within its SAND domain. Sp110 ubiquitination downregulates genes in the type I interferon response pathway. Comparative analysis of RNA-Seq from the UBR7/Sp110 knockdown data set confirmed that the IFN-β signaling pathway gets deregulated in HCC cells in the presence of HBV. Single-cell RNA-Seq analysis of patient samples further confirmed the inverse correlation between the expression of Sp110/UBR7 and the inflammation score. Notably, silencing of UBR7 induces IRF7 phosphorylation, thereby augmenting interferon (IFN)-β and the downstream interferon-stimulated genes (ISGs). Further, wild-type but not the ubiquitination-defective mutant of Sp110 could be recruited to the type I interferon response pathway genes. Our study establishes a new function of UBR7 in non-histone protein ubiquitination, promoting viral persistence, and has important implications for the development of therapeutic strategies targeting HBV-induced HCC.

Sirtuin 1 Dynamic Interplay of IFI16 and STING Interferon-Stimulated Gene Regulate Hepatitis B Virus Replication

Background: Hepatitis B virus (HBV) infection remains a significant global health concern, affecting millions worldwide and often leading to severe liver diseases. Chronic conditions associated with HBV contribute to the development of liver fibrosis, a crucial prognostic factor necessitating urgent therapeutic strategies. Objectives: This study aims to elucidate the interplay between interferon (IFN)-inducible protein 16 (IFI16), Sirtuin 1 (Sirt1) (NAD-dependent deacetylase), and STING (Stimulator of Interferon Genes) in the context of HBV infection, focusing on understanding their roles in viral replication and innate immune responses. Methods: This study investigates the interaction between Sirt1 and IFI16 during active HBV replication using siRNA-mediated knockdown and co-transfection techniques. HBV replication is assessed following IFI16 silencing, and the synergistic inhibition of IFI16 and Sirt1 is evaluated. Western blotting, electrophoresis, and immunoprecipitation methods are employed to explore STING's role in DNA-mediated innate immunity and interferon-stimulated gene activation during viral infection. Results: While individual knockdown of IFI16 has minimal impact on HBV replication, with a reduction of less than 10%, dual inhibition of IFI16 and Sirt1 resulted in a significant reduction in viral replication by approximately 70%. This underscores the synergistic role of these proteins in the context of HBV infection. Furthermore, the study implicates STING as a promising therapeutic target for viral infections, shedding light on its regulatory role in innate immune responses and interferon-stimulated genes (ISGs). Conclusions: Our study reveals the complex interplay between IFI16, Sirt1, and STING in HBV infection, highlighting potential therapeutic targets. While in vitro findings offer valuable insights, in vivo validation and further exploration of broader pathway interactions are essential. Future efforts should prioritize translating these findings into clinical applications for HBV treatment.

Invention and characterization of a systemically administered, attenuated and killed bacteria-based multiple immune receptor agonist for anti-tumor immunotherapy

Activation of immune receptors, such as Toll-like (TLR), NOD-like (NLR) and Stimulator of Interferon Genes (STING) is critical for efficient innate and adaptive immunity. Gram-negative bacteria (G-NB) contain multiple TLR, NOD and STING agonists. Potential utility of G-NB for cancer immunotherapy is supported by observations of tumor regression in the setting of infection and Coley’s Toxins. Coley reported that intravenous (i.v.) administration was likely most effective but produced uncontrollable toxicity. The discovery of TLRs and their agonists, particularly the potent TLR4 agonist lipopolysaccharide (LPS)-endotoxin, comprising ~75% of the outer membrane of G-NB, suggests that LPS may be both a critical active ingredient and responsible for dose-limiting i.v. toxicity of G-NB. This communication reports the production of killed, stabilized, intact bacteria products from non-pathogenic G-NB with ~96% reduction of LPS-endotoxin activity. One resulting product candidate, Decoy10, was resistant to standard methods of cell disruption and contained TLR2,4,8,9, NOD2 and STING agonist activity. Decoy10 also exhibited reduced i.v. toxicity in mice and rabbits, and a largely uncompromised ability to induce cytokine and chemokine secretion by human immune cells in vitro, all relative to unprocessed, parental bacterial cells. Decoy10 and a closely related product, Decoy20, produced single agent anti-tumor activity or combination-mediated durable regression of established subcutaneous, metastatic or orthotopic colorectal, hepatocellular (HCC), pancreatic, and non-Hodgkin’s lymphoma (NHL) tumors in mice, with induction of both innate and adaptive immunological memory (syngeneic and human tumor xenograft models). Decoy bacteria combination-mediated regressions were observed with a low-dose, oral non-steroidal anti-inflammatory drug (NSAID), anti-PD-1 checkpoint therapy, low-dose cyclophosphamide (LDC), and/or a targeted antibody (rituximab). Efficient tumor eradication was associated with plasma expression of 15-23 cytokines and chemokines, broad induction of cytokine, chemokine, innate and adaptive immune pathway genes in tumors, cold to hot tumor inflammation signature transition, and required NK, CD4+ and CD8+ T cells, collectively demonstrating a role for both innate and adaptive immune activation in the anti-tumor immune response.

The cGAS-STING pathway in HIV-1 and Mycobacterium tuberculosis coinfection.

Mycobacterium tuberculosis (M. tuberculosis) infection is the most common opportunistic infection in human immunodeficiency virus-1 (HIV-1)-infected individuals, and the mutual reinforcement of these two pathogens may accelerate disease progression and lead to rapid mortality. Therefore, HIV-1/M. tuberculosis coinfection is one of the major global public health concerns. HIV-1 infection is the greatest risk factor for M. tuberculosis infection and increases the likelihood of endogenous relapse and exogenous reinfection with M. tuberculosis. Moreover, M. tuberculosis further increases HIV-1 replication and the occurrence of chronic immune activation, accelerating the progression of HIV-1 disease. Exploring the pathogenesis of HIV-1/M. tuberculosis coinfections is essential for the development of novel treatments to reduce the global burden of tuberculosis. Innate immunity, which is the first line of host immune defense, plays a critical role in resisting HIV-1 and M. tuberculosis infections. The role of the cyclic guanosine monophosphate-adenosine monophosphate synthase (cGAS)-stimulator of interferon genes (STING) signaling pathway, which is a major DNA-sensing innate immune signaling pathway, in HIV-1 infection and M. tuberculosis infection has been intensively studied. This paper reviews the role of the cGAS-STING signaling pathway in HIV-1 infection and M. tuberculosis infection and discusses the possible role of this pathway in HIV-1/M. tuberculosis coinfection to provide new insight into the pathogenesis of HIV-1/M. tuberculosis coinfection and the development of novel therapeutic strategies.

A Single-Atom Mn/MoO3- x Nanoagonist for Cascade cGAS/STING Activation in Tumor-Specific Catalytic Metalloimmunotherapy.

The cyclic GMP-AMP synthase (cGAS)/stimulator of interferon genes (STING) pathway plays a crucial role in initiating anti-tumor immunity. Despite the development of various STING agonists, their effectiveness is often limited by suboptimal activation efficiency and poor sustainability. To address this, a Mn/MoO3- x nanoagonist featuring Mn single-atom sites is presented, designed for cascade cGAS/STING activation in tumor-specific catalytic metalloimmunotherapy. The single-atom nanoagonist (SANA) is meticulously crafted by doping Mn atoms into defective molybdenum oxide (MoO3- x), enabling robust peroxidase-mimicking catalysis and inducing severe double-stranded DNA (dsDNA) damage in tumors. Of note, Mn2+ and MoO4 2- can be responsively released from Mn/MoO3- x SANA and enhance the sensitivity of cGAS to dsDNA. Importantly, MoO4 2- with a relatively slow-release profile and facile cellular accumulation compensates for Mn2+ that has poor cellular accumulation due to continuous efflux, thus continuatively triggering the secretion of type I interferon for beyond 72 h. Remarkably, Mn/MoO3- x SANA significantly inhibits tumor growth and metastasis without supplementary STING agonists or external stimulation. This study offers a promising cascade cGAS/STING activation approach to enhance the efficacy and sustainability of catalytic metalloimmunotherapy.

PINK1-mediated mitophagy attenuates pathological cardiac hypertrophy by suppressing the mtDNA release-activated cGAS-STING pathway.

Sterile inflammation is implicated in the development of heart failure (HF). Mitochondria plays important roles in triggering and maintaining inflammation. Mitophagy is important for regulation of mitochondrial quality and maintenance of cardiac function under pressure overload. The association of mitophagy with inflammation in HF is largely unclear. As PINK1 is a central mediator of mitophagy, our objective was to investigate its involvement in cardiac hypertrophy, and the effect of PINK1-mediated mitophagy on cGAS-STING activation during cardiac hypertrophy. PINK1 knockout and cardiac-specific PINK1-overexpressing transgenic mice were created and subsequently subjected to transverse aortic constriction (TAC) surgery. In order to explore whether PINK1 regulates STING-mediated inflammation during HF, PINK1/STING (stimulator of interferon genes) double-knockout mice were created. Pressure overload was induced by TAC. Our findings indicate a significantly decline in PINK1 expression in TAC-induced hypertrophy. Cardiac hypertrophic stimuli caused the release of mitochondrial DNA (mtDNA) into the cytosol, activating the cGAS-STING signaling, which in turn initiated cardiac inflammation and promoted the progression of cardiac hypertrophy. PINK1 deficiency inhibited mitophagy activity, promoted mtDNA release, and then drove the overactivation of cGAS-STING signaling, exacerbating cardiac hypertrophy. Conversely, cardiac-specific PINK1 overexpression protected against hypertrophy thorough inhibition of the cGAS-STING signaling. Double-knockout mice revealed that the effects of PINK1 on hypertrophy were dependent on STING. Our findings suggest that PINK1-mediated mitophagy plays a protective role in pressure overload-induced cardiac hypertrophy via inhibiting the mtDNA-cGAS-STING pathway.

The long non-coding RNA lncRNA-DRNR enhances infectious bronchitis virus replication by targeting chicken JMJD6 and modulating interferon-stimulated genes expression via the JAK-STAT signalling pathway.

Infectious bronchitis virus (IBV) is the causative agent of infectious bronchitis (IB), a severe disease that primarily affects young chickens and poses a significant challenge to the global poultry industry. Understanding the complex interaction between the virus and its host is vital for developing innovative antiviral strategies. Long non-coding RNA (lncRNA) plays a crucial role in regulating host antiviral immune responses. Our previous studies have shown that IBV infection disrupts the stability of lncRNA in host cells, indicating a potential regulatory role for lncRNA in IBV pathogenesis. It is still not clear how lncRNA precisely modulates IBV replication. In this study, we observed down-regulation ofMSTRG.26120.58 (named lncRNA-DRNR) expression in various chicken cell lines upon IBV infection. We demonstrated that silencing lncRNA-DRNR using siRNA enhances intracellular replication of IBV. Through exploring genes encoding proteins upstream and downstream of lncRNA-DRNR within a 100kb range, we identified chJMJD6 (chicken JMJD6) as a potential target gene negatively regulated by lncRNA-DRNR expression levels. Furthermore, chJMJD6 inhibits STAT1 methylation, thereby affecting the induction of interferon-stimulated genes (ISGs) through the activation of the IFN-β-mediated JAK-STAT signalling pathway, ultimately promoting the intracellular replication of IBV. In summary, our findings reveal the critical role played by lncRNA-DRNR during IBV infection, providing novel insights into mechanisms underlying coronavirus-induced disruption in lncRNA stability.