The Ca2+-dependent phosphatase calcineurin dephosphorylates TBK1 to suppress antiviral innate immunity.

Tumor necrosis factor receptor-associated factor family member-associated NF-κB activator-binding kinase 1 (TBK1) plays a key role in the induction of the type 1 interferon (IFN-I) response, which is an important component of innate antiviral defense. Viruses target calcium (Ca2+) signaling networks, which participate in the regulation of the viral life cycle, as well as mediate the host antiviral response. Although many studies have focused on the role of Ca2+ signaling in the regulation of IFN-I, the relationship between Ca2+ and TBK1 in different infection models requires further elucidation. Here, we examined the effects of the Newcastle disease virus (NDV)-induced increase in intracellular Ca2+ levels on the suppression of host antiviral responses. We demonstrated that intracellular Ca2+ increased significantly during NDV infection, leading to impaired IFN-I production and antiviral immunity through the activation of calcineurin (CaN). Depletion of Ca²+ was found to lead to a significant increase in virus-induced IFN-I production resulting in the inhibition of viral replication. Mechanistically, the accumulation of Ca2+ in response to viral infection increases the phosphatase activity of CaN, which in turn dephosphorylates and inactivates TBK1 in a Ca2+-dependent manner. Furthermore, the inhibition of CaN on viral replication was counteracted in TBK1 knockout cells. Together, our data demonstrate that NDV hijacks Ca2+ signaling networks to negatively regulate innate immunity via the CaN-TBK1 signaling axis. Thus, our findings not only identify the mechanism by which viruses exploit Ca2+ signaling to evade the host antiviral response but also, more importantly, highlight the potential role of Ca2+ homeostasis in the viral innate immune response.IMPORTANCEViral infections disrupt intracellular Ca2+ homeostasis, which affects the regulation of various host processes to create conditions that are conducive for their own proliferation, including the host immune response. The mechanism by which viruses trigger TBK1 activation and IFN-I induction through viral pathogen-associated molecular patterns has been well defined. However, the effects of virus-mediated Ca2+ imbalance on the IFN-I pathway requires further elucidation, especially with respect to TBK1 activation. Herein, we report that NDV infection causes an increase in intracellular free Ca2+ that leads to activation of the serine/threonine phosphatase CaN, which subsequently dephosphorylates TBK1 and negatively regulates IFN-I production. Furthermore, depletion of Ca2+ or inhibition of CaN activity exerts antiviral effects by promoting the production of IFN-I and inhibiting viral replication. Thus, our results reveal the potential role of Ca2+ in the innate immune response to viruses and provide a theoretical reference for the treatment of viral infectious diseases.

Deep profiling of potential substrate atlas of porcine epidemic diarrhea virus 3C-like protease.

Coronavirus (CoV) 3C-like protease (3CLpro) is essential for viral replication and is involved in immune escape by proteolyzing host proteins. Deep profiling the 3CLpro substrates in the host proteome extends our understanding of viral pathogenesis and facilitates antiviral drug discovery. Here, 3CLpro from porcine epidemic diarrhea virus (PEDV), an enteropathogenic CoV, was used as a model which to identify the potential 3CLpro cleavage motifs in all porcine proteins. We characterized the selectivity of PEDV 3CLpro at sites P5-P4'. We then compiled the 3CLpro substrate preferences into a position-specific scoring matrix and developed a 3CLpro profiling strategy to delineate the protein substrate landscape of CoV 3CLpro. We identified 1,398 potential targets in the porcine proteome containing at least one putative cleavage site and experimentally validated the reliability of the substrate degradome. The PEDV 3CLpro-targeted pathways are involved in mRNA processing, translation, and key effectors of autophagy and the immune system. We also demonstrated that PEDV 3CLpro suppresses the type 1 interferon (IFN-I) cascade via the proteolysis of multiple signaling adaptors in the retinoic acid-inducible gene I (RIG-I) signaling pathway. Our composite method is reproducible and accurate, with an unprecedented depth of coverage for substrate motifs. The 3CLpro substrate degradome establishes a comprehensive substrate atlas that will accelerate the investigation of CoV pathogenicity and the development of anti-CoV drugs.IMPORTANCECoronaviruses (CoVs) are major pathogens that infect humans and animals. The 3C-like protease (3CLpro) encoded by CoV not only cleaves the CoV polyproteins but also degrades host proteins and is considered an attractive target for the development of anti-CoV drugs. However, the comprehensive characterization of an atlas of CoV 3CLpro substrates is a long-standing challenge. Using porcine epidemic diarrhea virus (PEDV) 3CLpro as a model, we developed a method that accurately predicts the substrates of 3CLpro and comprehensively maps the substrate degradome of PEDV 3CLpro. Interestingly, we found that 3CLpro may simultaneously degrade multiple molecules responsible for a specific function. For instance, it cleaves at least four adaptors in the RIG-I signaling pathway to suppress type 1 interferon production. These findings highlight the complexity of the 3CLpro substrate degradome and provide new insights to facilitate the development of anti-CoV drugs.

Dual fluorescence reporter mice for Ccl3 transcription, translation, and intercellular communication.

Chemokines guide immune cells during their response against pathogens and tumors. Various techniques exist to determine chemokine production, but none to identify cells that directly sense chemokines in vivo. We have generated CCL3-EASER (ErAse, SEnd, Receive) mice that simultaneously report for Ccl3 transcription and translation, allow identifying Ccl3-sensing cells, and permit inducible deletion of Ccl3-producing cells. We infected these mice with murine cytomegalovirus (mCMV), where Ccl3 and NK cells are critical defense mediators. We found that NK cells transcribed Ccl3 already in homeostasis, but Ccl3 translation required type I interferon signaling in infected organs during early infection. NK cells were both the principal Ccl3 producers and sensors of Ccl3, indicating auto/paracrine communication that amplified NK cell response, and this was essential for the early defense against mCMV. CCL3-EASER mice represent the prototype of a new class of dual fluorescence reporter mice for analyzing cellular communication via chemokines, which may be applied also to other chemokines and disease models.

Miz1 represses type I interferon production and limits viral clearance during influenza A virus infection.

Type I interferons (IFNs) are critical for the antiviral immune response, and fine-tuning type I IFN production is critical to effectively clearing viruses without causing harmful immunopathology. We showed that the transcription factor Miz1 epigenetically repressed the expression of genes encoding type I IFNs in mouse lung epithelial cells by recruiting histone deacetylase 1 (HDAC1) to the promoters of Ifna and Ifnb. Loss of function of Miz1 resulted in augmented production of these type I IFNs during influenza A virus (IAV) infection, leading to improved viral clearance in vitro and in vivo. IAV infection induced Miz1 accumulation by promoting the cullin-4B (CUL4B)-mediated ubiquitylation and degradation of the E3 ubiquitin ligase Mule (Mcl-1 ubiquitin ligase E3; also known as Huwe1 or Arf-BP1), which targets Miz1 for degradation. As a result, Miz1 accumulation limited type I IFN production and favored viral replication. This study reveals a previously unrecognized function of Miz1 in regulating antiviral defense and a potential mechanism for influenza viruses to evade host immune defense.

cGAS-STING pathway mediates activation of dendritic cell sensing of immunogenic tumors.

Type I interferons (IFN-I) play pivotal roles in tumor therapy for three decades, underscoring the critical importance of maintaining the integrity of the IFN-1 signaling pathway in radiotherapy, chemotherapy, targeted therapy, and immunotherapy. However, the specific mechanism by which IFN-I contributes to these therapies, particularly in terms of activating dendritic cells (DCs), remains unclear. Based on recent studies, aberrant DNA in the cytoplasm activates the cyclic GMP-AMP synthase (cGAS)- stimulator of interferon genes (STING) signaling pathway, which in turn produces IFN-I, which is essential for antiviral and anticancer immunity. Notably, STING can also enhance anticancer immunity by promoting autophagy, inflammation, and glycolysis in an IFN-I-independent manner. These research advancements contribute to our comprehension of the distinctions between IFN-I drugs and STING agonists in the context of oncology therapy and shed light on the challenges involved in developing STING agonist drugs. Thus, we aimed to summarize the novel mechanisms underlying cGAS-STING-IFN-I signal activation in DC-mediated antigen presentation and its role in the cancer immune cycle in this review.

Type I interferon regulates interleukin-1beta and IL-18 production and secretion in human macrophages.

Inflammasomes are immune complexes whose activation leads to the release of pro-inflammatory cytokines IL-18 and IL-1ß. Type I IFNs play a role in fighting infection and stimulate the expression of IFN-stimulated genes (ISGs) involved in inflammation. Despite the importance of these cytokines in inflammation, the regulation of inflammasomes by type I IFNs remains poorly understood. Here, we analysed RNA-sequencing data from patients with monogenic interferonopathies and found an up-regulation of several inflammasome-related genes. To investigate the effect of type I IFN on the inflammasome, we treated human monocyte-derived macrophages with IFN-α and observed an increase in CASP1 and GSDMD mRNA levels over time, whereas IL1B and NLRP3 were not directly correlated to IFN-α exposure time. IFN-α treatment reduced the release of mature IL-1ß and IL-18, but not caspase-1, in response to ATP-mediated NLRP3 inflammasome activation, suggesting regulation occurs at cytokine expression levels and not the inflammasome itself. However, more studies are required to investigate how regulation by IFN-α occurs and impacts NLRP3 and other inflammasomes at both transcriptional and post-translational levels.

Type I Interferon, Induced by Adenovirus or Adenoviral Vector Infection, Regulates the Cytokine Response to Lipopolysaccharide in a Macrophage Type-Specific Manner.

INTRODUCTION: While TLR ligands derived from microbial flora and pathogens are important activators of the innate immune system, a variety of factors such as intracellular bacteria, viruses, and parasites can induce a state of hyperreactivity, causing a dysregulated and potentially life-threatening cytokine over-response upon TLR ligand exposure. Type I interferon (IFN-αß) is a central mediator in the induction of hypersensitivity and is strongly expressed in splenic conventional dendritic cells (cDC) and marginal zone macrophages (MZM) when mice are infected with adenovirus. This study investigates the ability of adenoviral infection to influence the activation state of the immune system and underlines the importance of considering this state when planning the treatment of patients. METHODS: Infection with adenovirus-based vectors (Ad) or pretreatment with recombinant IFN-ß was used as a model to study hypersensitivity to lipopolysaccharide (LPS) in mice, murine macrophages, and human blood samples. The TNF-α, IL-6, IFN-αß, and IL-10 responses induced by LPS after pretreatment were measured. Mouse knockout models for MARCO, IFN-αßR, CD14, IRF3, and IRF7 were used to probe the mechanisms of the hypersensitive reaction. RESULTS: We show that, similar to TNF-α and IL-6 but not IL-10, the induction of IFN-αß by LPS increases strongly after Ad infection. This is true both in mice and in human blood samples ex vivo, suggesting that the regulatory mechanisms seen in the mouse are also present in humans. In mice, the scavenger receptor MARCO on IFN-αß-producing cDC and splenic marginal zone macrophages is important for Ad uptake and subsequent cytokine overproduction by LPS. Interestingly, not all IFN-αß-pretreated macrophage types exposed to LPS exhibit an enhanced TNF-α and IL-6 response. Pretreated alveolar macrophages and alveolar macrophage-like murine cell lines (MPI cells) show enhanced responses, while bone marrow-derived and peritoneal macrophages show a weaker response. This correlates with the respective absence or presence of the anti-inflammatory IL-10 response in these different macrophage types. In contrast, Ad or IFN-ß pretreatment enhances the subsequent induction of IFN-αß in all macrophage types. IRF3 is dispensable for the LPS-induced IFN-αß overproduction in infected MPI cells and partly dispensable in infected mice, while IRF7 is required. The expression of the LPS co-receptor CD14 is important but not absolutely required for the elicitation of a TNF-α over-response to LPS in Ad-infected mice. CONCLUSION: Viral infections or application of virus-based vaccines induces type I interferon and can tip the balance of the innate immune system in the direction of hyperreactivity to a subsequent exposure to TLR ligands. The adenoviral model presented here is one example of how multiple factors, both environmental and genetic, affect the physiological responses to pathogens. Being able to measure the current reactivity state of the immune system would have important benefits for infection-specific therapies and for the prevention of vaccination-elicited adverse effects.

Mechanisms of mesothelial cell response to viral infections: HDAC1-3 inhibition blocks poly(I:C)-induced type I interferon response and modulates the mesenchymal/inflammatory phenotype.

Infectious peritonitis is a leading cause of peritoneal functional impairment and a primary factor for therapy discontinuation in peritoneal dialysis (PD) patients. Although bacterial infections are a common cause of peritonitis episodes, emerging evidence suggests a role for viral pathogens. Toll-like receptors (TLRs) specifically recognize conserved pathogen-associated molecular patterns (PAMPs) from bacteria, viruses, and fungi, thereby orchestrating the ensuing inflammatory/immune responses. Among TLRs, TLR3 recognizes viral dsRNA and triggers antiviral response cascades upon activation. Epigenetic regulation, mediated by histone deacetylase (HDAC), has been demonstrated to control several cellular functions in response to various extracellular stimuli. Employing epigenetic target modulators, such as epidrugs, is a current therapeutic option in several cancers and holds promise in treating viral diseases. This study aims to elucidate the impact of TLR3 stimulation on the plasticity of human mesothelial cells (MCs) in PD patients and to investigate the effects of HDAC1-3 inhibition. Treatment of MCs from PD patients with the TLR3 agonist polyinosinic:polycytidylic acid (Poly(I:C)), led to the acquisition of a bona fide mesothelial-to-mesenchymal transition (MMT) characterized by the upregulation of mesenchymal genes and loss of epithelial-like features. Moreover, Poly(I:C) modulated the expression of several inflammatory cytokines and chemokines. A quantitative proteomic analysis of MCs treated with MS-275, an HDAC1-3 inhibitor, unveiled altered expression of several proteins, including inflammatory cytokines/chemokines and interferon-stimulated genes (ISGs). Treatment with MS-275 facilitated MMT reversal and inhibited the interferon signature, which was associated with reduced STAT1 phosphorylation. However, the modulation of inflammatory cytokine/chemokine production was not univocal, as IL-6 and CXCL8 were augmented while TNF-α and CXCL10 were decreased. Collectively, our findings underline the significance of viral infections in acquiring a mesenchymal-like phenotype by MCs and the potential consequences of virus-associated peritonitis episodes for PD patients. The observed promotion of MMT reversal and interferon response inhibition by an HDAC1-3 inhibitor, albeit without a general impact on inflammatory cytokine production, has translational implications deserving further analysis.

Aryl hydrocarbon receptor suppresses STING-mediated type I IFN expression in triple-negative breast cancer.

Triple-negative breast cancer (TNBC) is one of the most aggressive types of cancer. Despite decades of intense investigation, treatment options remain limited, and rapid recurrence with distant metastases remains a significant challenge. Cancer cell-intrinsic production of cytokines such as type I interferons (IFN-I) is a known potent modulator of response to therapy in many cancers, including TNBC, and can influence therapeutic outcome. Here, we report that, in TNBC systems, the aryl hydrocarbon receptor (AhR) suppresses IFN-I expression via inhibition of STImulator of Interferon Genes (STING), a key mediator of interferon production. Intratumoral STING activity is essential in mediating the efficacy of PARP inhibitors (PARPi) which are used in the treatment of cancers harboring BRCA1 deficiency. We find that, in TNBC cells, PARPi treatment activates AhR in a BRCA1 deficiency-dependent manner, thus suggesting the presence of a negative feedback loop aimed at modulating PARPi efficacy. Importantly, our results indicate that the combined inhibition of PARP and AhR is superior in elevating IFN-I expression as compared to PARPi-alone. Thus, AhR inhibition may allow for enhanced IFN-I production upon PARPi in BRCA1-deficient breast cancers, most of which are of TNBC origin, and may represent a therapeutically viable strategy to enhance PARPi efficacy.

VRK1 promotes DNA-induced type I interferon production.

BACKGROUND: Type I interferons (IFNs) are an essential class of cytokines with antitumor, antiviral and immunoregulatory effects. However, over-productive the type I IFNs are tightly associated with autoimmune disorders. Thus, the induction of type I interferons is precisely regulated to maintain immune hemostasis. This study aimed to identify a novel regulator of type I interferon signaling. METHODS AND RESULTS: Primary BMDMs, isolated from mice, and human cell lines (HEK293 cells, Hela cells) and murine cell line (MEF cells) were cultured to generate in vitro models. After knockdown VRK1, real-time PCR and dual-luciferase reporter assay were performed to determine the expression level of the type I IFNs and ISGs following HTDNA and Poly (dA:dT) stimulation. Additionally, cells were treated with the VRK1 inhibitor, and the impact of VRK1 inhibition was detected. Upon HTDNA and Poly (dA:dT) stimulation, knockdown of VRK1 attenuated the induction of the type I IFNs and ISGs. Consistently, VRK-IN-1, a potent and selective VRK1 inhibitor, significantly suppressed the induction of the type I IFNs and ISGs in human and murine cell lines. Further, VRK-IN-1 inhibited induction of the type I IFNs in mouse primary BMDMs. Intriguingly, VRK1 potentiated the cGAS-STING- IFN-I axis response at STING level. CONCLUSIONS: Our study reveals a novel function of VRK1 in regulating the production of type I IFNs. VRK-IN-1 might be a potential lead compound for suppressing aberrant type I IFNs in autoimmune disorders.

STAT3 protects hematopoietic stem cells by preventing activation of a deleterious autocrine type-I interferon response.

Hematopoietic stem and progenitor cells (HSPCs) maintain blood-forming and immune activity, yet intrinsic regulators of HSPCs remain elusive. STAT3 function in HSPCs has been difficult to dissect as Stat3-deficiency in the hematopoietic compartment induces systemic inflammation, which can impact HSPC activity. Here, we developed mixed bone marrow (BM) chimeric mice with inducible Stat3 deletion in 20% of the hematopoietic compartment to avoid systemic inflammation. Stat3-deficient HSPCs were significantly impaired in reconstitution ability following primary or secondary bone marrow transplantation, indicating hematopoietic stem cell (HSC) defects. Single-cell RNA sequencing of Lin-ckit+Sca1+ BM cells (LSKs) revealed aberrant activation of cell cycle, p53, and interferon (IFN) pathways in Stat3-deficient HSPCs. Stat3-deficient LSKs accumulated γH2AX and showed increased expression of DNA sensors and type-I IFN (IFN-I), while treatment with A151-ODN inhibited expression of IFN-I and IFN-responsive genes. Further, the blockade of IFN-I receptor signaling suppressed aberrant cell cycling, STAT1 activation, and nuclear p53 accumulation. Collectively, our results show that STAT3 inhibits a deleterious autocrine IFN response in HSCs to maintain long-term HSC function. These data signify the importance of ensuring therapeutic STAT3 inhibitors are targeted specifically to diseased cells to avoid off-target loss of healthy HSPCs.

The tumor cell-intrinsic cGAS-STING pathway is associated with the high density of CD8+ T cells after chemotherapy in esophageal squamous cell carcinoma.

BACKGROUND: Chemotherapy has the potential to induce CD8+ T-cell infiltration in the tumor microenvironment (TME) and activate the anti-tumor immune response in several cancers including esophageal squamous cell carcinoma (ESCC). The tumor cell-intrinsic cyclic GMP-AMP synthase (cGAS)-stimulator of interferon genes (STING) pathway has been known as a critical component for regulating immune cell activation in the TME. However, its effect on the infiltration of immune cells induced by chemotherapy in the ESCC TME has not been investigated. METHODS: We examined the effect of the tumor-cell intrinsic cGAS-STING pathway on the infiltration of CD8+ T cells induced by chemotherapy in ESCC using ESCC cell lines and surgically resected ESCC specimens from patients who received neoadjuvant chemotherapy (NAC). RESULTS: We found that chemotherapeutic agents, including 5-fluorouracil (5-FU) and cisplatin (CDDP), activated the cGAS-STING pathway, consequently inducing the expression of type I interferon and T-cell-attracting chemokines in ESCC cells. Moreover, the tumor cell-intrinsic expression of cGAS-STING was significantly and positively associated with the density of CD8+ T cells in ESCC after NAC. However, the tumor cell-intrinsic expression of cGAS-STING did not significantly impact clinical outcomes in patients with ESCC after NAC. CONCLUSION: Our findings suggest that the tumor cell-intrinsic cGAS-STING pathway might contribute to chemotherapy-induced immune cell activation in the ESCC TME.

STING licensing of type I dendritic cells potentiates antitumor immunity.

Stimulator of interferon genes (STING) is an immune adaptor protein that senses cyclic GMP-AMP in response to self or microbial cytosolic DNA as a danger signal. STING is ubiquitously expressed in diverse cell populations, including cancer cells, with distinct cellular functions, such as activation of type I interferons, autophagy induction, or triggering apoptosis. It is not well understood whether and which subsets of immune cells, stromal cells, or cancer cells are particularly important for STING-mediated antitumor immunity. Here, using a polymeric STING-activating nanoparticle (PolySTING) with a shock-and-lock dual activation mechanism, we show that conventional type 1 dendritic cells (cDC1s) are essential for STING-mediated rejection of multiple established and metastatic murine tumors. STING status in the host but not in the cancer cells (Tmem173-/-) is important for antitumor efficacy. Specific depletion of cDC1 (Batf3-/-) or STING deficiency in cDC1 (XCR1creSTINGfl/fl) abolished PolySTING efficacy, whereas depletion of other myeloid cells had little effect. Adoptive transfer of wild-type cDC1 in Batf3-/- mice restored antitumor efficacy, whereas transfer of cDC1 with STING or IRF3 deficiency failed to rescue. PolySTING induced a specific chemokine signature in wild-type but not Batf3-/- mice. Multiplexed immunohistochemistry analysis of STING-activating cDC1s in resected tumors correlates with patient survival. Furthermore, STING-cDC1 signature was increased after neoadjuvant pembrolizumab therapy in patients with non-small cell lung cancer. Therefore, we have defined that a subset of myeloid cells is essential for STING-mediated antitumor immunity with associated biomarkers for prognosis.

The gut microbiota contributes to the infection of bovine viral diarrhea virus in mice.

Bovine viral diarrhea virus (BVDV) is prevalent worldwide and causes significant economic losses. Gut microbiota is a large microbial community and has a variety of biological functions. However, whether there is a correlation between gut microbiota and BVDV infection and what kind of relation between them have not been reported. Here, we found that gut microbiota composition changed in normal mice after infecting with BVDV, but mainly the low abundance microbe was affected. Interestingly, BVDV infection significantly reduced the diversity of gut microbiota and changed its composition in gut microbiota-dysbiosis mice. Furthermore, compared with normal mice of BVDV infection, there were more viral loads in the duodenum, jejunum, spleen, and liver of the gut microbiota-dysbiosis mice. However, feces microbiota transplantation (FMT) reversed these effects. The data above indicated that the dysbiosis of gut microbiota was a key factor in the high infection rate of BVDV. It is found that the IFN-I signal was involved by investigating the underlying mechanisms. The inhibition of the proliferation and increase in the apoptosis of peripheral blood lymphocytes (PBL) were also observed. However, FMT treatment reversed these changes by regulating PI3K/Akt, ERK, and Caspase-9/Caspase-3 pathways. Furthermore, the involvement of butyrate in the pathogenesis of BVDV was also further confirmed. Our results showed for the first time that gut microbiota acts as a key endogenous defense mechanism against BVDV infection; moreover, targeting regulation of gut microbiota structure and abundance may serve as a new strategy to prevent and control the disease.IMPORTANCEWhether the high infection rate of BVDV is related to gut microbiota has not been reported. In addition, most studies on BVDV focus on in vitro experiments, which limits the study of its prevention and control strategy and its pathogenic mechanism. In this study, we successfully confirmed the causal relationship between gut microbiota and BVDV infection as well as the potential molecular mechanism based on a mouse model of BVDV infection and a mouse model of gut microbiota dysbiosis. Meanwhile, a mouse model which is more susceptible to BVDV provided in this study lays an important foundation for further research on prevention and control strategy of BVDV and its pathogenesis. In addition, the antiviral effect of butyrate, the metabolites of butyrate-producing bacteria, has been further revealed. Overall, our findings provide a promising prevention and control strategy to treat this infectious disease which is distributed worldwide.

Inhibition of Aurora Kinase Induces Endogenous Retroelements to Induce a Type I/III IFN Response via RIG-I.

Type I IFN signaling is a crucial component of antiviral immunity that has been linked to promoting the efficacy of some chemotherapeutic drugs. We developed a reporter system in HCT116 cells that detects activation of the endogenous IFI27 locus, an IFN target gene. We screened a library of annotated compounds in these cells and discovered Aurora kinase inhibitors (AURKi) as strong hits. Type I IFN signaling was found to be the most enriched gene signature after AURKi treatment in HCT116, and this signature was also strongly enriched in other colorectal cancer cell lines. The ability of AURKi to activate IFN in HCT116 was dependent on MAVS and RIG-I, but independent of STING, whose signaling is deficient in these cells. MAVS dependence was recapitulated in other colorectal cancer lines with STING pathway deficiency, whereas in cells with intact STING signaling, the STING pathway was required for IFN induction by AURKi. AURKis were found to induce expression of endogenous retroviruses (ERV). These ERVs were distinct from those induced by the DNA methyltransferase inhibitors (DNMTi), which can induce IFN signaling via ERV induction, suggesting a novel mechanism of action. The antitumor effect of alisertib in mice was accompanied by an induction of IFN expression in HCT116 or CT26 tumors. CT26 tumor growth inhibition by alisertib was absent in NSG mice versus wildtype (WT) mice, and tumors from WT mice with alisertib treatment showed increased in CD8+ T-cell infiltration, suggesting that antitumor efficacy of AURKi depends, at least in part, on an intact immune response. SIGNIFICANCE: Some cancers deactivate STING signaling to avoid consequences of DNA damage from aberrant cell division. The surprising activation of MAVS/RIG-I signaling by AURKi might represent a vulnerability in STING signaling deficient cancers.

Dual-Epigenetically Relieving the MYC-Correlated Immunosuppression via an Advanced Nano-Radiosensitizer Potentiates Cancer Immuno-Radiotherapy.

Cancer cells can upregulate the MYC expression to repair the radiotherapy-triggered DNA damage, aggravating therapeutic resistance and tumor immunosuppression. Epigenetic treatment targeting the MYC-transcriptional abnormality may intensively solve this clinical problem. Herein, 5-Aza (a DNA methyltransferase inhibitor) and ITF-2357 (a histone deacetylase inhibitor) are engineered into a tungsten-based nano-radiosensitizer (PWAI), to suppress MYC rising and awaken robust radiotherapeutic antitumor immunity. Individual 5-Aza depletes MYC expression but cannot efficiently awaken radiotherapeutic immunity. This drawback can be overcome by the addition of ITF-2357, which triggers cancer cellular type I interferon (IFN-I) signaling. Coupling 5-Aza with ITF-2357 ensures that PWAI does not evoke the treated model with high MYC-related immune resistance while amplifying the radiotherapeutic tumor killing, and more importantly promotes the generation of IFN-I signal-related proteins involving IFN-α and IFN-ß. Unlike the radiation treatment alone, PWAI-triggered immuno-radiotherapy remarkably enhances antitumor immune responses involving the tumor antigen presentation by dendritic cells, and improves intratumoral recruitment of cytotoxic T lymphocytes and their memory-phenotype formation in 4T1 tumor-bearing mice. Downgrading the radiotherapy-induced MYC overexpression via the dual-epigenetic reprogramming strategy may elicit a robust immuno-radiotherapy.

Targeting PRMT9-mediated arginine methylation suppresses cancer stem cell maintenance and elicits cGAS-mediated anticancer immunity.

Current anticancer therapies cannot eliminate all cancer cells, which hijack normal arginine methylation as a means to promote their maintenance via unknown mechanisms. Here we show that targeting protein arginine N-methyltransferase 9 (PRMT9), whose activities are elevated in blasts and leukemia stem cells (LSCs) from patients with acute myeloid leukemia (AML), eliminates disease via cancer-intrinsic mechanisms and cancer-extrinsic type I interferon (IFN)-associated immunity. PRMT9 ablation in AML cells decreased the arginine methylation of regulators of RNA translation and the DNA damage response, suppressing cell survival. Notably, PRMT9 inhibition promoted DNA damage and activated cyclic GMP-AMP synthase, which underlies the type I IFN response. Genetically activating cyclic GMP-AMP synthase in AML cells blocked leukemogenesis. We also report synergy of a PRMT9 inhibitor with anti-programmed cell death protein 1 in eradicating AML. Overall, we conclude that PRMT9 functions in survival and immune evasion of both LSCs and non-LSCs; targeting PRMT9 may represent a potential anticancer strategy.

Role of micronucleus-activated cGAS-STING signaling in antitumor immunity. / å¾®æ ¸æ¿€æ´»cGAS-STINGä¿¡å·é€šè·¯çš„æœºåˆ¶åŠå ¶è‚¿ç˜¤å ç–«åŠŸèƒ½.

Cyclic guanosine monophosphate-adenosine monophosphate synthase (cGAS)-stimulator of interferon genes (STING) signaling is a significant component of the innate immune system and functions as a vital sentinel mechanism to monitor cellular and tissue aberrations in microbial invasion and organ injury. cGAS, a cytosolic DNA sensor, is specialized in recognizing abnormally localized cytoplasmic double-stranded DNA (dsDNA) and catalyzes the formation of a second messenger cyclic-GMP-AMP (cGAMP), which initiates a cascade of type Ⅰ interferon and inflammatory responses mediated by STING. Micronucleus, a byproduct of chromosomal missegregation during anaphase, is also a significant contributor to cytoplasmic dsDNA. These unstable subcellular structures are susceptible to irreversible nuclear envelope rupture, exposing genomic dsDNA to the cytoplasm, which potently recruits cGAS and activates STING-mediated innate immune signaling and its downstream activities, including type Ⅰ interferon and classical nuclear factor-κB (NF-κB) signaling pathways lead to senescence, apoptosis, autophagy activating anti-cancer immunity or directly killing tumor cells. However, sustained STING activation-induced endoplasmic reticulum stress, activated chronic type Ⅰ interferon and nonclassical NF-κB signaling pathways remodel immunosuppressive tumor microenvironment, leading to immune evasion and facilitating tumor metastasis. Therefore, activated cGAS-STING signaling plays a dual role of suppressing or facilitating tumor growth in tumorigenesis and therapy. This review elaborates on research advances in mechanisms of micronucleus inducing activation of cGAS-STING signaling and its implications in tumorigenesis and therapeutic strategies of malignant tumors.

MicroRNA-218-5p-Ddx41 axis restrains microglia-mediated neuroinflammation through downregulating type I interferon response in a mouse model of Parkinson's disease.

BACKGROUND: Parkinson's disease (PD) is a neurodegenerative disorder characterized by the loss of dopaminergic (DA) neurons in the substantia nigra (SN). Microglia-mediated neuroinflammation has been largely considered one of main factors to the PD pathology. MicroRNA-218-5p (miR-218-5p) is a microRNA that plays a role in neurodevelopment and function, while its potential function in PD and neuroinflammation remains unclear. METHODS: We explore the involvement of miR-218-5p in the PD in a 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine (MPTP)-induced mouse model. The miR-218-5p agomir used for overexpression was delivered into the substantia nigra (SN) by bilateral stereotaxic infusions. The loss of dopaminergic (DA) neurons and microglial inflammation in the SN was determined using Western blotting and immunofluorescence. Motor function was assessed using the rotarod test. RNA sequencing (RNA-seq) was performed to explore the pathways regulated by miR-218-5p. The target genes of miR-218-5p were predicted using TargetScan and confirmed using dual luciferase reporter assays. The effects of miR-218-5p on microglial inflammation and related pathways were verified in murine microglia-like BV2 cells. To stimulate BV2 cells, SH-SY5Y cells were treated with 1-methyl-4-phenylpyridinium (MPP+) and the conditioned media (CM) were collected. RESULTS: MiR-218-5p expression was reduced in both the SN of MPTP-induced mice and MPP+-treated BV2 cells. MiR-218-5p overexpression significantly alleviated MPTP-induced microglial inflammation, loss of DA neurons, and motor dysfunction. RNA sequence and gene set enrichment analysis showed that type I interferon (IFN-I) pathways were upregulated in MPTP-induced mice, while this upregulation was reversed by miR-218-5p overexpression. A luciferase reporter assay verified that Ddx41 was a target gene of miR-218-5p. In vitro, miR-218-5p overexpression or Ddx41 knockdown inhibited the IFN-I response and expression of inflammatory cytokines in BV2 cells stimulated with MPP+-CM. CONCLUSIONS: MiR-218-5p suppresses microglia-mediated neuroinflammation and preserves DA neurons via Ddx41/IFN-I. Hence, miR-218-5p-Ddx41 is a promising therapeutic target for PD.

Enterovirus D68 3C protease antagonizes type I interferon signaling by cleaving signal transducer and activator of transcription 1.

Following the successful control of poliovirus, the re-emergence of respiratory enterovirus D68 (EV-D68), a prominent non-polio enterovirus, has become a serious public health concern worldwide. Host innate immune responses are the primary defense against EV-D68 invasion; however, the mechanism underlying viral evasion of the antiviral activity of interferons (IFN) remains unclear. In this study, we found that EV-D68 inhibited type I IFN signaling by cleaving signal transducer and activator of transcription 1 (STAT1), a crucial factor in cellular responses to interferons and other cytokines. We observed that the prototype and circulating EV-D68 strains conserved their ability to induce STAT1 cleavage and attenuate IFN signal transduction. Further investigation revealed that EV-D68 3C protease cleaves STAT1 at the 131Q residue. Interestingly, not all enterovirus-encoded 3C proteases exhibited this ability. EV-D68 and poliovirus 3C proteases efficiently induced STAT1 cleavage; whereas, 3C proteases from EV-A71, coxsackievirus A16, and echoviruses did not. STAT1 cleavage also abolished the nuclear translocation capacity of STAT1 in response to IFN stimulation to activate downstream signaling elements. Overall, these results suggest that STAT1, targeted by viral protease 3C, is utilized by EV-D68 to subvert the host's innate immune response.IMPORTANCEEnterovirus D68 (EV-D68) has significantly transformed over the past decade, evolving from a rare pathogen to a potential pandemic pathogen. The interferon (IFN) signaling pathway is an important defense mechanism and therapeutic target for the host to resist viral invasion. Previous studies have reported that the EV-D68 virus blocks or weakens immune recognition and IFN production in host cells through diverse strategies; however, the mechanisms of EV-D68 resistance to IFN signaling have not been fully elucidated. Our study revealed that EV-D68 relies on its own encoded protease, 3C, to directly cleave signal transducer and activator of transcription 1 (STAT1), a pivotal transduction component in the IFN signaling pathway, disrupting the IFN-mediated antiviral response. Previous studies on human enteroviruses have not documented direct cleavage of the STAT1 protein to evade cellular immune defenses. However, not all enteroviral 3C proteins can cleave STAT1. These findings highlight the diverse evolutionary strategies different human enteroviruses employ to evade host immunity.