TANK-binding Kinase Research Articles

Cyclic stretch regulates immune responses via tank-binding kinase 1 expression in macrophages.

Macrophages distributed in tissues throughout the body contribute to homeostasis. In the inflammatory state, macrophages undergo mechanical stress that regulates the signal transduction of immune responses and various cellular functions. However, the effects of the inflammatory response on macrophages under physiological cyclic stretch are unclear. We found that physiological cyclic stretch suppresses inflammatory cytokine expression in macrophages by regulating NF-κB activity. NF-κB phosphorylation at Ser536 in macrophages was inhibited, suggesting that tank-binding kinase (TBK1) regulates NF-κB activity during physiological stress. Moreover, TBK1 expression was suppressed by physiological stretch, and TBK1 knockdown by siRNA induced the suppression of NF-κB phosphorylation at Ser536. In conclusion, physiological stretch triggers suppression of a TBK1-dependent excessive inflammatory response, which may be necessary to maintain tissue homeostasis.

Mutational analysis and protein profiling predict drug sensitivity in multiple myeloma cell lines.

Multiple myeloma (MM) is a heterogeneous disease where cancer-driver mutations and aberrant signaling may lead to disease progression and drug resistance. Drug responses vary greatly, and there is an unmet need for biomarkers that can guide precision cancer medicine in this disease. To identify potential predictors of drug sensitivity, we applied integrated data from drug sensitivity screening, mutational analysis and functional signaling pathway profiling in 9 cell line models of MM. We studied the sensitivity to 33 targeted drugs and their association with the mutational status of cancer-driver genes and activity level of signaling proteins. We found that sensitivity to mitogen-activated protein kinase kinase 1 (MEK1) and phosphatidylinositol-3 kinase (PI3K) inhibitors correlated with mutations in NRAS/KRAS, and PI3K family genes, respectively. Phosphorylation status of MEK1 and protein kinase B (AKT) correlated with sensitivity to MEK and PI3K inhibition, respectively. In addition, we found that enhanced phosphorylation of proteins, including Tank-binding kinase 1 (TBK1), as well as high expression of B cell lymphoma 2 (Bcl-2), correlated with low sensitivity to MEK inhibitors. Taken together, this study shows that mutational status and signaling protein profiling might be used in further studies to predict drug sensitivities and identify resistance markers in MM.

Hereditary glaucoma: clinical and genetic characteristics

The review is devoted to the genetic nature of congenital glaucoma (CG) and presents clinical and genetic forms of hereditary glaucoma and single nucleotide polymorphisms identified by genome-wide association studies (GWAS). Glaucoma is a genetically heterogeneous disease, and patients with the same clinical diagnosis often have different molecular causes. The role of mutations in the CYP1B1 gene has been proven in the pathogenesis of hydrophthalmos; the MYOC gene — in juvenile open-angle glaucoma; the PAX6 gene — in aniridia; mutations in the PITX2, FOXC1 genes have been identified in Axenfeld-Rieger anomaly/syndrome. It has been established that 4–43% of patients with open-angle glaucoma have a family history of a mutation in the MYOC, OPTN or TBK1 genes. Genetic studies of glaucoma are the first steps to developing a new generation of personalized treatments. The article describes the key features of the pathogenesis of various genetic forms of glaucoma and the possible course of its therapy. However, gene therapy requires further study of both long-term effects and efficacy. Molecular genetic diagnosis of glaucoma allows for personalized genetic counseling of family members with consideration of the genetic risks.

The Porcine Cyclic GMP-AMP Synthase-STING Pathway Exerts an Unusual Antiviral Function Independent of Interferon and Autophagy.

The innate immune DNA-sensing cyclic GMP-AMP synthase (cGAS)-stimulator of interferon (IFN) gene (STING) pathway exerts strong antiviral activity through downstream IFN production; however, it has been recently recognized that an IFN-independent activity of STING also plays an important role in antiviral functions. Nevertheless, the IFN-independent antiviral activity of STING is not fully understood. Here, we showed that porcine STING (pSTING) played a critical role against herpes simplex virus 1 (HSV-1) and vesicular stomatitis virus (VSV) infections, and IFN-defective mutants, including pSTING pLxIS sub, S365A, and △CTT, all exhibited similar antiviral functions, compared to wild-type (WT) pSTING. Furthermore, all of these IFN-defective pSTING mutants possessed comparable autophagy activity, relative to WT pSTING, as expected. From pSTING WT, S365A, and △CTT, the residues responsible for autophagy, including L333A/R334A, Y167A/L170A, and Y245A/L248A, were mutated. Surprisingly, all of these autophagy-defective pSTING mutants still resisted the two viral infections, demonstrating that the pSTING antiviral function is independent of IFN as well as autophagy. On the other hand, all of the autophagy-defective pSTING mutants triggered cell apoptosis, which was associated with and participated in the antiviral functions. Additionally, pSTING lost its antiviral activity in TANK-binding kinase 1 (TBK1)-/- and IFN regulatory factor 3 (IRF3)-/- porcine macrophages, indicating the involvement of TBK1 and IRF3 in other STING activities such as apoptosis. Collectively, our results revealed that STING exerts both IFN- and autophagy-independent antiviral activity, and they also suggested that STING-triggered cell apoptosis resists viral infections. IMPORTANCE The IFN-independent antiviral function of the cGAS-STING pathway has attracted great attention in recent years; however, the nature of this IFN-independent antiviral function is unknown, although STING-induced autophagy has been shown to mediate the STING antiviral activity. First, we analyzed the antiviral activity through the porcine cGAS-pSTING pathway and established that pSTING signaling exerts an IFN-independent antiviral function. Second, we found that pSTING-induced IFN-independent autophagy and the antiviral activity of pSTING are independent of both IFN and autophagy. Finally, pSTING signaling activates cell apoptosis independently of IFN and autophagy, and the apoptosis is associated with antiviral activity. Our results suggest that pSTING-activated apoptosis at least partially mediates the antiviral activity or multiple pSTING-activated signals, including IFN production, nuclear factor κ light chain enhancer of activated B cells (NF-κB) expression, autophagy, and apoptosis, exert a redundant antiviral role. Thus, the work reveals a new layer of complexity in STING antiviral activity.

Analysis of COVID-19-Associated Proteins Identifies TANK-Binding Kinase 1 As an Immunotherapeutic Target in Multiple Myeloma

Background and Rationale. Patients with multiple myeloma (MM) are at a higher risk of viral infection due to immune deficiency, as recently highlighted by the severity of COVID-19 in MM patients. However, the mechanism(s) underlying the lack of anti-viral immune response in MM are unclear. We have previously shown that there are more plasmacytoid dendritic cells (pDCs) in the bone marrow of MM patients than in the bone marrow of healthy individuals and that these dysfunctional patient pDCs suppress T and NK cell-mediated immunity. Importantly, pDCs play a key role in both recognizing viral nucleic acid and initiating anti-viral immunity via type 1 interferon (IFN) response signaling. Therefore, we analyzed the immune-pathway proteins implicated in COVID-19-host interactions [COVID-19 protein interaction map, Gordon et al, Nature 2020; 583, 459-568] to assess whether pDC-MM interactions modulate these pathways to confer immune suppression and susceptibility to COVID-19. Results. We first assessed whether the expression of human genes involved in the COVID-19-host interaction changes when ex vivo MM cells are co-cultured with patient pDCs (compared to MM cells alone). We found that 41 of these genes were differentially expressed in MM cells when exposed to pDCs (log2Fold change: ± 3.5-fold; p < 0.00001), and this expression varied widely (median (log10): -0.13 to 4.5; adj p < 0.001). Three of these genes are linked to innate immunity and are essential for IFN production in MM cells - TBK1, IRF3 (Interferon regulatory factor 3), and RAE1 (interferon-inducible mRNA nuclear export factor). Importantly, pDCs downregulated the expression of TBK1 (Log2FC: -0.5) and RAE1 (Log2FC: -0.3) and upregulated the expression of IRF3 (Log2FC: 1.5) in MM cells after coculture (compared to MM cells alone, p < 0.0001). Concordantly, analysis of publicly available gene profiling datasets on relapsed MM patients showed low levels of TBK1 and RAE1 and higher levels of IRF3 (n = 50) (Log2FC: TBK1: -0.208; RAE1: -0.286; IRF3: 0.273; vs normal; p < 0.05). Of note, TBK1 expression was positively correlated with prognosis (n = 350, p = 0.026). We found that most of the 41 genes were also expressed in pDCs (NCBI GEO Datasets based on pDC cell line CAL1 and human peripheral blood pDCs). In unstimulated pDCs, TBK1 expression was significantly lower than IRF3 and RAE1 expression (2-3-fold, p < 0.05). Yet treating pDCs with CpG-ODN type-A, which induces IFN-α expression, increased both TBK1 (adj P = 0.004) and RAE1 (adj P = 0.043) expression, without significantly altering IRF3 expression. Finally, COVID-19 relies on the host ubiquitin-proteasome system for propagation, and we found that targeting ubiquitin receptor Rpn13 with the specific inhibitor RA190 restored TBK1 expression in MM cell lines, indicating the potential clinical use of targeting Rpn13 in restoring anti-viral immune responses in MM. Conclusion. Taken together, we show that TBK1 is downregulated in pDCs, and pDCs interacting with MM cells further decrease TBK1 expression in MM cells, thereby attenuating IFN-mediated anti-viral immune response signaling. Our findings suggest that: 1) low levels of TBK1 may confer increased susceptibility of MM patients to COVID-19 and 2) targeting TBK1 may restore the anti-viral immune response and reduce COVID-19 severity in MM.

IMMU-10. EXPLORING THE IMMUNOLOGIC CONSEQUENCES OF ATRX DEFICIENCY IN GLIOMA

Abstract ATRX is a key chromatin regulator, which is mutated in large subsets of both adult and pediatric gliomas. Despite being a common mutation, little is known about the biological ramifications of ATRX deficiency. Recently, it has been demonstrated that ATRX deficiency drives increased replication stress, DNA damage, and global epigenetic dysregulation. Despite these advances, little is known about the impact of ATRX deficiency on the tumor microenvironment (TME). In order to explore the impact of ATRX deficiency on the TME we utilized the RCAS/nTVa system to generate a novel murine model of ATRX deficient glioma. Mice bearing allografts of these tumors displayed significantly increased survival relative to the ATRX intact lines. This survival benefit persisted in Nu/nu mice, which lack an adaptive immune system, but not in SCID mice, which lack both adaptive and innate immunity. Bulk RNA sequencing revealed the ATRX deficient tumors displayed increased expression of inflammatory and innate immune gene sets, and western blotting revealed increased phosphorylation of tank binding kinase 1 (TBK1), a key innate immune regulator. Multiplex cytokine analysis of conditioned media also revealed increased expression of inflammatory cytokines such as CCL2, CCL5, and CXCL10 in the supernatant of ATRX deficient cell lines. Taken together, these results indicate that ATRX deficiency can drive innate immune activation, and that this activation can increase survival in vivo. Further exploration is needed to characterize the upstream drivers of TBK1 activation and elucidate alterations to immune cell infiltration in ATRXdeficient tumors.

Integrative Strategy for Investigating the Interactions between STING and Small-Molecule Ligands.

Although small-molecule agonists of stimulator of interferon genes (STING) show significance in activating the immune system, the dynamic process involved in ligands activating STING remains unclear. Herein, we developed a biochemical strategy, integrating computer simulation and a biochemical engineering approach, to reveal the interaction mechanism between STING and 5,6-dimethylxanthenone-4-acetic acid (DMXAA), an agonist that activates the TANK binding kinase 1-interferon regulatory factor 3 signaling pathway. Specifically, inspired by an analysis of the STING-DMXAA crystal structure, we designed and synthesized DMXAA derivatives to investigate the STING-DMXAA binding model. We identified that the carboxyl moiety of DMXAA was a major pharmacophore responsive to STING activation. In particular, the loss of hydrogen bond interaction between the carboxylic acid of DMXAA and the side chain Thr262 of STING led to STING inhibition. DMXAA N-methyl amide derivative (DNHM) exhibited good inhibitor activity, inhibited STING-mediated interferon production in vitro and in vivo, and effectively attenuated STING-associated inflammatory diseases. Therefore, we provide a new insight into STING-ligand interactions, which may improve the understanding of STING biology.

Abstract 9345: Midline1 is a Critical Regulator of Viral Myocarditis

Introduction: Infections by viruses including severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) could cause viral myocarditis. Innate immunity to viral nucleic acids mediates antiviral immunity as well as inflammatory organ injury. However, the innate immune mechanisms that control the pathogenesis of viral myocarditis are unclear. Methods: To understand the role of the E3 ligase Midline1 in controlling viral myocarditis, wild-type and Midline1 knockout mice were infected with coxsackievirus B3 (CVB3) or encephalomyocarditis (EMCV) for inducing viral myocarditis. Mice survivals and heart functions were monitored by transthoracic echocardiography, and hearts were harvested for histology analysis. Real-time PCR, western blotting, co-immunoprecipitation, enzyme-linked immunoassay, luciferase assay, flow cytometry, over-expression and knockdown techniques were used to understand the molecular mechanisms of Midline1 in regulating type I interferon production after virus infection in this study. Results: We find that Midline1 is induced by RNA viruses with heart tropisms CVB3 and EMCV. Knockdown or deletion of Midline1 in human or mouse macrophages enhances production of type I interferon (IFN) in response to double strand (ds) RNA, RNA viruses CVB3 or EMCV. Importantly, deletion of Midline1 protects mice from viral myocarditis induced by CVB3 or EMCV due to enhanced type I IFN production in vivo. Mechanistically, we show that Midline1 recruits protein phosphatase 1A (PPM1A) to dephosphorylate TANK binding kinase 1 (TBK1), which inactivates TBK1 to block TBK1 from interacting with its upstream adaptor mitochondrial antiviral signaling (MAVS), thereby dampening antiviral signaling during viral infections. Moreover, Midline1 stabilizes PPM1A by inducing K63-linked ubiquitination of PPM1A. Conclusions: Our results indicate that Midline1 serves as a negative regulator of viral myocarditis by downregulating innate immune activation induced by RNA viruses CVB3 and EMCV. Our data reveal that Midline1 is a critical regulator of innate immunity in viral myocarditis, thereby identifying a potential therapeutic target for treating viral induced cardiovascular diseases.

SRA Suppresses Antiviral Innate Immune Response in Macrophages by Limiting TBK1 K63 Ubiquitination via Deubiquitinase USP15.

The innate immune system is the first line of host defense against microbial infections. During virus infection, pattern recognition receptors (PRRs) are engaged to detect specific viral components, such as viral RNA or DNA, and regulate the innate immune response in the infected cells or immune cells. Our previous study demonstrated that scavenger receptor A (SRA), an important innate PRR, impaired the anti-hepatitis B virus (HBV) response in hepatocytes. Given that SRA is primarily expressed in macrophages, here, we assessed the function of SRA expressed in macrophages in response to RNA or DNA viral infection. SRA-deficient (SRA-/-) mice showed reduced susceptibility to viral infection caused by vesicular stomatitis virus (VSV) or herpes simplex virus 1 (HSV-1). In the virus-infected SRA-/- mice, compared with their wild-type (WT) counterparts, we observed low amounts of virus accompanied by enhanced interferon (IFN) production. Furthermore, SRA significantly inhibited the phosphorylation of TANK-binding kinase 1 (TBK1) and interferon regulatory factor 3 (IRF3). We provided biochemical evidence showing that SRA directly interacts with the N-terminal kinase domain (KD) of TBK1, resulting in the limitation of its K63-linked ubiquitination. Moreover, we demonstrated that SRA negatively regulates the activity of TBK1 by promoting the recruitment of ubiquitin-specific protease 15 (USP15) to deubiquitinate TBK1. In summary, we have identified the connection between SRA and the TBK1/IRF3 signaling pathway in macrophages, indicating a critical role of SRA in the regulation of host antiviral immunity. IMPORTANCE During virus infection, PRRs are engaged to detect specific viral components, such as viral RNA or DNA, and regulate the innate immune response in the infected cells or other immune cells. We reported that deficiency of SRA, an important innate PRR, promoted IRF3 activation, type I IFN production, and innate antiviral responses against RNA and DNA viruses in vivo and in vitro. Furthermore, the biochemical analysis showed that SRA directly interacts with the KD domain of TBK1 and limits its K63-linked polyubiquitination, reducing TBK1 activation. Further analyses determined that SRA is a modulator for TBK1 activation via the recruitment of USP15, which delineated a previously unrecognized function for SRA in innate antiviral immunity.

IRF5 knockdown reverses TDP-related phenotypes partially by increasing TBK1 expression

Interferon-regulatory factor 5 (IRF5) participates in the regulation of apoptosis, affects the phenotype of inflammatory macrophages and plays an essential role in the inflammatory response. However, the role of IRF5 in the progression of amyotrophic lateral sclerosis (ALS) remains largely unknown. Here, we show that IRF5 mainly accumulated in the nucleus in cells expressing the truncated 25 kD C-terminal fragments of TDP-43 (TDP-25, named TDP-25 cells hereafter). IRF5 knockdown using a lentivirus carrying an shRNA in TDP-25 cells exerted a protective effect and reduced the level of the apoptosis-related protein cleaved caspase-9 and the cell cycle arrest protein p21, while increasing the expression of the antioxidant transcription factor nuclear factor erythroid 2-related factor 2 (Nrf2) and its target molecule glutamate-cysteine ligase modulatory subunit (GCLM). Furthermore, IRF5-knockdown cells showed improved mitochondrial swelling and cristae dilation. In addition, we found that IRF5 mediated neuronal injury partly through the negative regulation of TANK-binding kinase 1 (TBK1). These data indicate that the loss of IRF5 in TDP-25 cells exerts a protective effect mainly by inhibiting apoptosis, regulating cell cycle arrest and alleviating oxidative stress.

TBK1 inhibitors enhance transfection efficiency by suppressing p62/SQSTM1 phosphorylation.

DNA transfection is an essential technique in the life sciences. Non-viral transfection reagents are widely used for transfection in basic science. However, low transfection efficiency is a problem in some cell types. This low efficiency can be primarily attributed to the intracellular degradation of transfected DNA by p62-dependent selective autophagy, specifically by p62 phosphorylated at the S403 residue (p62-S403-P). To achieve efficient DNA transfection, we focused on a phosphorylation process that generates p62-S403-P and investigated whether inhibition of this process affects transfection efficiency. One of the kinases that phosphorylate p62 is TBK1. The TBK1 gene depletion in murine embryonic fibroblast cells by genome editing caused a significant reduction or loss of p62-S405-P (equivalent to human S403-P) and enhanced transfection efficiency, suggesting that TBK1 is a major kinase that phosphorylates p62 at S403. Therefore, TBK1 is a viable target for drug treatment to increase transfection efficiency. Transfection efficiency was enhanced when cells were treated with one of the following TBK1 inhibitors BX795, MRT67307, or amlexanox. This effect was synergistically improved when the two inhibitors were used in combination. Our results indicate that TBK1 inhibitors enhanced transfection efficiency by suppressing p62 phosphorylation.

Tripartite motif-containing protein 46 accelerates influenza A H7N9 virus infection by promoting K48-linked ubiquitination of TBK1

BackgroundAvian influenza A H7N9 emerged in 2013, threatening public health and causing acute respiratory distress syndrome, and even death, in the human population. However, the underlying mechanism by which H7N9 virus causes human infection remains elusive.MethodsHerein, we infected A549 cells with H7N9 virus for different times and assessed tripartite motif-containing protein 46 (TRIM46) expression. To determine the role of TRIM46 in H7N9 infection, we applied lentivirus-based TRIM46 short hairpin RNA sequences and overexpression plasmids to explore virus replication, and changes in type I interferons and interferon regulatory factor 3 (IRF3) phosphorylation levels in response to silencing and overexpression of TRIM46. Finally, we used Co-immunoprecipitation and ubiquitination assays to examine the mechanism by which TRIM46 mediated the activity of TANK-binding kinase 1 (TBK1).ResultsType I interferons play an important role in defending virus infection. Here, we found that TRIM46 levels were significantly increased during H7N9 virus infection. Furthermore, TRIM46 knockdown inhibited H7N9 virus replication compared to that in the control group, while the production of type I interferons increased. Meanwhile, overexpression of TRIM46 promoted H7N9 virus replication and decrease the production of type I interferons. In addition, the level of phosphorylated IRF3, an important interferon regulatory factor, was increased in TRIM46-silenced cells, but decreased in TRIM46 overexpressing cells. Mechanistically, we observed that TRIM46 could interact with TBK1 to induce its K48-linked ubiquitination, which promoted H7N9 virus infection.ConclusionOur results suggest that TRIM46 negatively regulates the human innate immune response against H7N9 virus infection.

Ubiquitin-specific protease 24 promotes EV71 infection by restricting K63-linked polyubiquitination of TBK1

TANK-binding kinase 1 (TBK1) is an essential protein kinase for activation of interferon regulatory factor 3 (IRF3) and induction of the type I interferons (IFN-I). Although the biochemical regulation of TBK1 activation has been studied, little is known about how enterovirus 71 (EV71) employs the deubiquitinases (DUBs) to regulate TBK1 activation for viral immune evasion. Here, we found that EV71 infection upregulated the expression of ubiquitin-specific protease 24 (USP24). Further studies revealed that USP24 physically interacted with TBK1, and can reduce K63-linked polyubiquitination of TBK1. Knockdown of USP24 upregulated TBK1 K63-linked polyubiquitination, promoted the phosphorylation and nuclear translocation of IRF3, and in turn improved IFN-I production during EV71 infection. As a consequence, USP24 knockdown dramatically inhibited EV71 infection. This study revealed USP24 as a novel regulator of TBK1 activation, which promotes the understanding of immune evasion mechanisms of EV71 and could provide a potential strategy for treatment of EV71 infection.

Enhanced IFN Sensing by Aggressive Chronic Lymphocytic Leukemia Cells.

Type I IFN is made by cells in response to stress. Cancer cells exist in a state of stress, but their IFN response is complex and not completely understood. This study investigated the role of autocrine IFN in human chronic lymphocytic leukemia (CLL) cells. CLL cells were found to make low amounts of IFN via TANK-binding kinase 1 pathways, but p-STAT1 and -STAT2 proteins along with IFN-stimulated genes that reflect IFN activation were variably downregulated in cultured CLL cells by the neutralizing IFNAR1 Ab anifrolumab. Patients with CLL were segregated into two groups based on the response of their leukemia cells to anifrolumab. Samples associated with more aggressive clinical behavior indicated by unmutated IGHV genes along with high CD38 and p-Bruton's tyrosine kinase expression exhibited responses to low amounts of IFN that were blocked by anifrolumab. Samples with more indolent behavior were unaffected by anifrolumab. Hypersensitivity to IFN was associated with higher expression of IFNAR1, MX1, STAT1, and STAT2 proteins and lower activity of negative regulatory tyrosine phosphatases. Autocrine IFN protected responsive CLL cells from stressful tissue culture environments and therapeutic drugs such as ibrutinib and venetoclax in vitro, in part by upregulating Mcl-1 expression. These findings suggest hypersensitivity to IFN may promote aggressive clinical behavior. Specific blockade of IFN signaling may improve outcomes for patients with CLL with higher-risk disease.

Let them eat virus: exploring how TBK1 (TANK binding kinase 1) enhances autophagic flux to promote autophagic degradation of coxsackievirus B

Coxsackievirus B (CVB) is a common human enterovirus that can cause an array of systemic inflammatory diseases. We and others have demonstrated that macroautophagy/autophagy is activated during CVB infection leading to viral engulfment within autophagosomes. Interestingly, rather than this mechanism leading to bulk degradation of intracellular virus (also referred to as xenophagy), autophagosome-lysosomal fusion appears to be circumvented, leading to extracellular release of CVB via ejected autophagosomes. In our present study, we have found that TBK1 (TANK binding kinase 1) plays a role in limiting CVB infection by promoting autophagic flux to limit autophagy-based viral egress. This aspect of viral defense also appears to be independent of TBK1’s well-characterized involvement in interferon signaling. Indeed, genetic inhibition of TBK1 significantly enhances CVB infection in vitro and dramatically increases the amount of vesicle-bound virus being released from the cell. Furthermore, inhibition of TBK1 via amlexanox treatment markedly increases serum levels of infectious extracellular vesicles (EV) and severity of pancreatitis in CVB-infected mice. In all, the identification of TBK1’s involvement in the suppression of CVB egress pegs it as a promising therapeutic target for the development of novel antiviral strategies against not only CVB but potentially other viruses that exploit autophagy to promote viral spread.

Constitutively active STING causes neuroinflammation and degeneration of dopaminergic neurons in mice.

Stimulator of interferon genes (STING) is activated after detection of cytoplasmic dsDNA by cGAS (cyclic GMP-AMP synthase) as part of the innate immunity defence against viral pathogens. STING binds TANK-binding kinase 1 (TBK1). TBK1 mutations are associated with familial amyotrophic lateral sclerosis, and the STING pathway has been implicated in the pathogenesis of further neurodegenerative diseases. To test whether STING activation is sufficient to induce neurodegeneration, we analysed a mouse model that expresses the constitutively active STING variant N153S. In this model, we focused on dopaminergic neurons, which are particularly sensitive to stress and represent a circumscribed population that can be precisely quantified. In adult mice expressing N153S STING, the number of dopaminergic neurons was smaller than in controls, as was the density of dopaminergic axon terminals and the concentration of dopamine in the striatum. We also observed alpha-synuclein pathology and a lower density of synaptic puncta. Neuroinflammation was quantified by staining astroglia and microglia, by measuring mRNAs, proteins and nuclear translocation of transcription factors. These neuroinflammatory markers were already elevated in juvenile mice although at this age the number of dopaminergic neurons was still unaffected, thus preceding the degeneration of dopaminergic neurons. More neuroinflammatory markers were blunted in mice deficient for inflammasomes than in mice deficient for signalling by type I interferons. Neurodegeneration, however, was blunted in both mice. Collectively, these findings demonstrate that chronic activation of the STING pathway is sufficient to cause degeneration of dopaminergic neurons. Targeting the STING pathway could therefore be beneficial in Parkinson's disease and further neurodegenerative diseases.

SARS-CoV-2, HIV, and HPV: Convergent evolution of selective regulation of cGAS-STING signaling.

Recognizing aberrant cytoplasmic double-stranded DNAand stimulating innate immunity is essential for the host's defense against viruses and tumors. Cyclic GMP-AMP (cGAMP) synthase (cGAS) is a cytosolic DNA sensor that synthesizes the second messenger 2'3'-cGAMP and subsequently activates stimulator of interferon genes (STING)-mediated activation of TANK-binding kinase 1 (TBK1)/interferon regulatory factor 3 (IRF3) and the production of type I interferon(IFN-I). Both the cGAS-STING-mediated IFN-I antiviral defense and the countermeasures developed by diverse viruses have been extensively studied. However, recent studies have revealed a convergent evolutionary feature of severe acute respiratory syndrome coronavirus 2and human immunodeficiency virus(HIV) viral proteins in terms of the selective regulation of cGAS-STING-mediated nuclear factor-κB (NF-κB) signaling without any effect on cGAS-STING-mediated TBK1/IRF3 activation and IFN production. The potential beneficial effect of this cGAS-STING-mediated, NF-κB-dependent antiviral effect, and the possible detrimental effect of IFN-I in the pathogenesis of coronavirus disease 2019and HIV infection deserve more attention and future investigation.

Chicken anemia virus VP1 negatively regulates type I interferon via targeting interferon regulatory factor 7 of the DNA-sensing pathway

The cyclic GMP-AMP synthase (cGAS)-stimulator of interferon genes (STING) signaling pathway plays a vital role in sensing viral DNA in the cytosol, stimulating type I interferon (IFN) production and triggering the innate immune response against DNA virus infection. However, viruses have evolved effective inhibitors to impede this sensing pathway. Chicken anemia virus (CAV), a nonenveloped ssDNA virus, is a ubiquitous pathogen causing great economic losses to the poultry industry globally. CAV infection is reported to downregulate type I IFN induction. However, whether the cGAS-STING signal axis is used by CAV to regulate type I IFN remains unclear. Our results demonstrate that CAV infection significantly elevates the expression of cGAS and STING at the mRNA level, whereas IFN-β levels are reduced. Furthermore, IFN-β activation was completely blocked by the structural protein VP1 of CAV in interferon stimulatory DNA (ISD) or STING-stimulated cells. VP1 was further confirmed as an inhibitor by interacting with interferon regulatory factor 7 (IRF7) by binding its C-terminal 143–492 aa region. IRF7 dimerization induced by TANK binding kinase 1 (TBK1) could be inhibited by VP1 in a dose-dependent manner. Together, our study demonstrates that CAV VP1 is an effective inhibitor that interacts with IRF7 and antagonizes cGAS-STING pathway-mediated IFN-β activation. These findings reveal a new mechanism of immune evasion by CAV.

Glycolaldehyde-derived advanced glycation end products suppress STING/TBK1/IRF3 signaling via CD36

AimsWe have previously reported that advanced glycation end products derived from incubation of albumin with glycolaldehyde (glycol-AGE), lead to suppression of the toll-like receptor 4 (TLR4) signaling response to lipopolysaccharide. Glycol-AGE-induced suppression of TLR4 signaling is involved in the downregulation of CD14, which is an adaptor protein necessary for transferring lipopolysaccharide to TLR4. Therefore, glycol-AGEs impair the innate immune response through suppression of the upstream process in TLR4 signaling. However, the effect of glycol-AGEs on intracellular signaling related to the innate immune response remains unclear. This study aimed to examined the effect of glycol-AGEs on stimulator of interferon gene (STING) signaling in macrophages. Main methodsIn differentiated THP-1 cells, which are a human monocytic leukemia cell line, cyclic GMP-AMP (cGAMP) transfection was used to activate STING signaling. The phosphorylation levels of TANK-binding kinase 1 (TBK1)/interferon regulatory transcription factor 3 (IRF3) were evaluated by western blot analysis. Downstream cytokine levels were evaluated by quantitative real-time polymerase chain reaction and enzyme-linked immunosorbent assays. Key findingsGlycol-AGEs suppressed cGAMP-induced phosphorylation of TBK1 and IRF3, as well as the production of cytokines regulated by IRF3. There was no effect of glycol-AGEs on the efficacy of cGAMP transfection. Treatment of a neutralizing antibody against CD36 prevented cGAMP-induced phosphorylation of TBK1 and IRF3, and also upregulation of interferon-β and C-X-C motif chemokine ligand 10 in glycol-AGE-treated cells. SignificanceGlycol-AGEs negatively regulate cGAMP-induced activation of STING/TBK1/IRF3 signaling via CD36. Our findings suggest that glycol-AGEs lead to impairment of the innate immune response by suppressing intracellular signaling.

Inosine: A broad-spectrum anti-inflammatory against SARS-CoV-2 infection-induced acute lung injury via suppressing TBK1 phosphorylation

Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2)-induced cytokine storms constitute the primary cause of coronavirus disease 19 (COVID-19) progression, severity, criticality, and death. Glucocorticoid and anti-cytokine therapies are frequently administered to treat COVID-19, but have limited clinical efficacy in severe and critical cases. Nevertheless, the weaknesses of these treatment modalities have prompted the development of anti-inflammatory therapy against this infection. We found that the broad-spectrum anti-inflammatory agent inosine downregulated proinflammatory interleukin (IL)-6, upregulated anti-inflammatory IL-10, and ameliorated acute inflammatory lung injury caused by multiple infectious agents. Inosine significantly improved survival in mice infected with SARS-CoV-2. It indirectly impeded TANK-binding kinase 1 (TBK1) phosphorylation by binding stimulator of interferon genes (STING) and glycogen synthase kinase-3β (GSK3β), inhibited the activation and nuclear translocation of the downstream transcription factors interferon regulatory factor (IRF3) and nuclear factor kappa B (NF-κB), and downregulated IL-6 in the sera and lung tissues of mice infected with lipopolysaccharide (LPS), H1N1, or SARS-CoV-2. Thus, inosine administration is feasible for clinical anti-inflammatory therapy against severe and critical COVID-19. Moreover, targeting TBK1 is a promising strategy for inhibiting cytokine storms and mitigating acute inflammatory lung injury induced by SARS-CoV-2 and other infectious agents.