An IFNγ-dependent immune-endocrine circuit lowers blood glucose to potentiate the innate antiviral immune response.

Viral infection makes us feel sick as the immune system alters systemic metabolism to better fight the pathogen. The extent of these changes is relative to the severity of disease. Whether blood glucose is subject to infection-induced modulation is mostly unknown. Here we show that strong, nonlethal infection restricts systemic glucose availability, which promotes the antiviral type I interferon (IFN-I) response. Following viral infection, we find that IFNγ produced by γδ T cells stimulates pancreatic ß cells to increase glucose-induced insulin release. Subsequently, hyperinsulinemia lessens hepatic glucose output. Glucose restriction enhances IFN-I production by curtailing lactate-mediated inhibition of IRF3 and NF-κB signaling. Induced hyperglycemia constrained IFN-I production and increased mortality upon infection. Our findings identify glucose restriction as a physiological mechanism to bring the body into a heightened state of responsiveness to viral pathogens. This immune-endocrine circuit is disrupted in hyperglycemia, possibly explaining why patients with diabetes are more susceptible to viral infection.

Deactylation by SIRT1 enables liquid-liquid phase separation of IRF3/IRF7 in innate antiviral immunity.

Innate antiviral immunity deteriorates with aging but how this occurs is not entirely clear. Here we identified SIRT1-mediated DNA-binding domain (DBD) deacetylation as a critical step for IRF3/7 activation that is inhibited during aging. Viral-stimulated IRF3 underwent liquid-liquid phase separation (LLPS) with interferon (IFN)-stimulated response element DNA and compartmentalized IRF7 in the nucleus, thereby stimulating type I IFN (IFN-I) expression. SIRT1 deficiency resulted in IRF3/IRF7 hyperacetylation in the DBD, which inhibited LLPS and innate immunity, resulting in increased viral load and mortality in mice. By developing a genetic code expansion orthogonal system, we demonstrated the presence of an acetyl moiety at specific IRF3/IRF7 DBD site/s abolish IRF3/IRF7 LLPS and IFN-I induction. SIRT1 agonists rescued SIRT1 activity in aged mice, restored IFN signaling and thus antagonized viral replication. These findings not only identify a mechanism by which SIRT1 regulates IFN production by affecting IRF3/IRF7 LLPS, but also provide information on the drivers of innate immunosenescence.

The Cytoplasmic DNA Sensor cGAS Promotes Mitotic Cell Death.

Pathogenic and other cytoplasmic DNAs activate the cyclic GMP-AMP synthase (cGAS)-stimulator of interferon genes (STING) pathway to induce inflammation via transcriptional activation by IRF3 and nuclear factor κB (NF-κB), but the functional consequences of exposing cGAS to chromosomes upon mitotic nuclear envelope breakdown are unknown. Here, we show that nucleosomes competitively inhibit DNA-dependent cGAS activation and that the cGAS-STING pathway is not effectively activated during normal mitosis. However, during mitotic arrest, low level cGAS-dependent IRF3 phosphorylation slowly accumulates without triggering inflammation. Phosphorylated IRF3, independently of its DNA-binding domain, stimulates apoptosis through alleviating Bcl-xL-dependent suppression of mitochondrial outer membrane permeabilization. We propose that slow accumulation of phosphorylated IRF3, normally not sufficient for inducing inflammation, can trigger transcription-independent induction of apoptosis upon mitotic aberrations. Accordingly, expression of cGAS and IRF3 in cancer cells makes mouse xenograft tumors responsive to the anti-mitotic agent Taxol. The Cancer Genome Atlas (TCGA) datasets for non-small cell lung cancer patients also suggest an effect of cGAS expression on taxane response.

Cyclic GMP-AMP as an Endogenous Second Messenger in Innate Immune Signaling by Cytosolic DNA.

The innate immune system functions as the first line of defense against invading bacteria and viruses. In this context, the cGAS/STING [cyclic guanosine monophosphate (GMP)-adenosine monophosphate (AMP) synthase/STING] signaling axis perceives the nonself DNA associated with bacterial and viral infections, as well as the leakage of self DNA by cellular dysfunction and stresses, to elicit the host's immune responses. In this pathway, the noncanonical cyclic dinucleotide 2',3'-cyclic GMP-AMP (2',3'-cGAMP) functions as a second messenger for signal transduction: 2',3'-cGAMP is produced by the enzyme cGAS upon its recognition of double-stranded DNA, and then the 2',3'-cGAMP is recognized by the receptor STING to induce the phosphorylation of downstream factors, including TBK1 (TANK binding kinase 1) and IRF3 (interferon regulatory factor 3). Numerous crystal structures of the components of this cGAS/STING signaling axis have been reported and these clarify the structural basis for their signal transduction mechanisms. In this review, we summarize recent progress made in the structural dissection of this signaling pathway and indicate possible directions of forthcoming research.

Micronuclei induced by radiation, replication stress, or chromosome segregation errors do not activate cGAS-STING.

The cyclic guanosine monophosphate (GMP)-AMP synthase (cGAS)-stimulator of interferon genes (STING) pathway plays a pivotal role in innate immune responses to viral infection and inhibition of autoimmunity. Recent studies have suggested that micronuclei formed by genotoxic stress can activate innate immune signaling via the cGAS-STING pathway. Here, we investigated cGAS localization, activation, and downstream signaling from micronuclei induced by ionizing radiation, replication stress, and chromosome segregation errors. Although cGAS localized to ruptured micronuclei via binding to self-DNA, we failed to observe cGAS activation; cGAMP production; downstream phosphorylation of STING, TBK1, or IRF3; nuclear accumulation of IRF3; or expression of interferon-stimulated genes. Failure to activate the cGAS-STING pathway was observed across primary and immortalized cell lines, which retained the ability to activate the cGAS-STING pathway in response to dsDNA or modified vaccinia virus infection. We provide evidence that micronuclei formed by genotoxic insults contain histone-bound self-DNA, which we show is inhibitory to cGAS activation in cells.

RNA-binding protein isoforms ZAP-S and ZAP-L have distinct antiviral and immune resolution functions.

The initial response to viral infection is anticipatory, with host antiviral restriction factors and pathogen sensors constantly surveying the cell to rapidly mount an antiviral response through the synthesis and downstream activity of interferons. After pathogen clearance, the host's ability to resolve this antiviral response and return to homeostasis is critical. Here, we found that isoforms of the RNA-binding protein ZAP functioned as both a direct antiviral restriction factor and an interferon-resolution factor. The short isoform of ZAP bound to and mediated the degradation of several host interferon messenger RNAs, and thus acted as a negative feedback regulator of the interferon response. In contrast, the long isoform of ZAP had antiviral functions and did not regulate interferon. The two isoforms contained identical RNA-targeting domains, but differences in their intracellular localization modulated specificity for host versus viral RNA, which resulted in disparate effects on viral replication during the innate immune response.

Vaginal Exposure to Zika Virus during Pregnancy Leads to Fetal Brain Infection.

Zika virus (ZIKV) can be transmitted sexually between humans. However, it is unknown whether ZIKV replicates in the vagina and impacts the unborn fetus. Here, we establish a mouse model of vaginal ZIKV infection and demonstrate that, unlike other routes, ZIKV replicates within the genital mucosa even in wild-type (WT) mice. Mice lacking RNA sensors or transcription factors IRF3 and IRF7 resulted in higher levels of local viral replication. Furthermore, mice lacking the type I interferon (IFN) receptor (IFNAR) became viremic and died of infection after a high-dose vaginal ZIKV challenge. Notably, vaginal infection of pregnant dams during early pregnancy led to fetal growth restriction and infection of the fetal brain in WT mice. This was exacerbated in mice deficient in IFN pathways, leading to abortion. Our study highlights the vaginal tract as a highly susceptible site of ZIKV replication and illustrates the dire disease consequences during pregnancy.

Metabolic enzyme UAP1 mediates IRF3 pyrophosphorylation to facilitate innate immune response.

Post-translational modifications (PTMs) of proteins are crucial to guarantee the proper biological functions in immune responses. Although protein phosphorylation has been extensively studied, our current knowledge of protein pyrophosphorylation, which occurs based on phosphorylation, is very limited. Protein pyrophosphorylation is originally considered to be a non-enzymatic process, and its function in immune signaling is unknown. Here, we identify a metabolic enzyme, UDP-N-acetylglucosamine pyrophosphorylase 1 (UAP1), as a pyrophosphorylase for protein serine pyrophosphorylation, by catalyzing the pyrophosphorylation of interferon regulatory factor 3 (IRF3) at serine (Ser) 386 to promote robust type I interferon (IFN) responses. Uap1 deficiency significantly impairs the activation of both DNA- and RNA-viruse-induced type I IFN pathways, and the Uap1-deficient mice are highly susceptible to lethal viral infection. Our findings demonstrate the function of protein pyrophosphorylation in the regulation of antiviral responses and provide insights into the crosstalk between metabolism and innate immunity.

YAP antagonizes innate antiviral immunity and is targeted for lysosomal degradation through IKKɛ-mediated phosphorylation.

The transcription regulator YAP controls organ size by regulating cell growth, proliferation and apoptosis. However, whether YAP has a role in innate antiviral immunity is largely unknown. Here we found that YAP negatively regulated an antiviral immune response. YAP deficiency resulted in enhanced innate immunity, a diminished viral load, and morbidity in vivo. YAP blocked dimerization of the transcription factor IRF3 and impeded translocation of IRF3 to the nucleus after viral infection. Notably, virus-activated kinase IKKɛ phosphorylated YAP at Ser403 and thereby triggered degradation of YAP in lysosomes and, consequently, relief of YAP-mediated inhibition of the cellular antiviral response. These findings not only establish YAP as a modulator of the activation of IRF3 but also identify a previously unknown regulatory mechanism independent of the kinases Hippo and LATS via which YAP is controlled by the innate immune pathway.

NLRX1 promotes immediate IRF1-directed antiviral responses by limiting dsRNA-activated translational inhibition mediated by PKR.

NLRX1 is unique among the nucleotide-binding-domain and leucine-rich-repeat (NLR) proteins in its mitochondrial localization and ability to negatively regulate antiviral innate immunity dependent on the adaptors MAVS and STING. However, some studies have suggested a positive regulatory role for NLRX1 in inducing antiviral responses. We found that NLRX1 exerted opposing regulatory effects on viral activation of the transcription factors IRF1 and IRF3, which might potentially explain such contradictory results. Whereas NLRX1 suppressed MAVS-mediated activation of IRF3, it conversely facilitated virus-induced increases in IRF1 expression and thereby enhanced control of viral infection. NLRX1 had a minimal effect on the transcription of IRF1 mediated by the transcription factor NF-kB and regulated the abundance of IRF1 post-transcriptionally by preventing translational shutdown mediated by the double-stranded RNA (dsRNA)-activated kinase PKR and thereby allowed virus-induced increases in the abundance of IRF1 protein.

Decreased Expression of the Host Long-Noncoding RNA-GM Facilitates Viral Escape by Inhibiting the Kinase activity TBK1 via S-glutathionylation.

Viruses have evolved multiple strategies to evade elimination by the immune system. Here we examined the contribution of host long noncoding RNAs (lncRNAs) in viral immune evasion. By functional screening of lncRNAs whose expression decreased upon viral infection of macrophages, we identified a lncRNA (lncRNA-GM, Gene Symbol: AK189470.1) that promoted type I interferon (IFN-I) production and inhibited viral replication. Deficiency of lncRNA-GM in mice increased susceptibility to viral infection and impaired IFN-I production. Mechanistically, lncRNA-GM bound to glutathione S-transferase M1 (GSTM1) and blocked GSTM1 interaction with the kinase TBK1, reducing GSTM1-mediated S-glutathionylation of TBK1. Decreased S-glutathionylation enhanced TBK1 activity and downstream production of antiviral mediators. Viral infection reprogrammed intracellular glutathione metabolism and furthermore, an oxidized glutathione mimetic could inhibit TBK1 activity and promote viral replication. Our findings reveal regulation of TBK1 by S-glutathionylation and provide insight into the viral mediated metabolic changes that impact innate immunity and viral evasion.

Targeting 7-Dehydrocholesterol Reductase Integrates Cholesterol Metabolism and IRF3 Activation to Eliminate Infection.

Recent work suggests that cholesterol metabolism impacts innate immune responses against infection. However, the key enzymes or the natural products and mechanisms involved are not well elucidated. Here, we have shown that upon DNA and RNA viral infection, macrophages reduced 7-dehydrocholesterol reductase (DHCR7) expression. DHCR7 deficiency or treatment with the natural product 7-dehydrocholesterol (7-DHC) could specifically promote phosphorylation of IRF3 (not TBK1) and enhance type I interferon (IFN-I) production in macrophages. We further elucidated that viral infection or 7-DHC treatment enhanced AKT3 expression and activation. AKT3 directly bound and phosphorylated IRF3 at Ser385, together with TBK1-induced phosphorylation of IRF3 Ser386, to achieve IRF3 dimerization. Deletion of DHCR7 and the DHCR7 inhibitors including AY9944 and the chemotherapy drug tamoxifen promoted clearance of Zika virus and multiple viruses in vitro or in vivo. Taken together, we propose that the DHCR7 inhibitors and 7-DHC are potential therapeutics against emerging or highly pathogenic viruses.

Induced phase separation of mutant NF2 imprisons the cGAS-STING machinery to abrogate antitumor immunity.

Missense mutations of the tumor suppressor Neurofibromin 2 (NF2/Merlin/schwannomin) result in sporadic to frequent occurrences of tumorigenesis in multiple organs. However, the underlying pathogenicity of NF2-related tumorigenesis remains mostly unknown. Here we found that NF2 facilitated innate immunity by regulating YAP/TAZ-mediated TBK1 inhibition. Unexpectedly, patient-derived individual mutations in the FERM domain of NF2 (NF2m) converted NF2 into a potent suppressor of cGAS-STING signaling. Mechanistically, NF2m gained extreme associations with IRF3 and TBK1 and, upon innate nucleic acid sensing, was directly induced by the activated IRF3 to form cellular condensates, which contained the PP2A complex, to eliminate TBK1 activation. Accordingly, NF2m robustly suppressed STING-initiated antitumor immunity in cancer cell-autonomous and -nonautonomous murine models, and NF2m-IRF3 condensates were evident in human vestibular schwannomas. Our study reports phase separation-mediated quiescence of cGAS-STING signaling by a mutant tumor suppressor and reveals gain-of-function pathogenesis for NF2-related tumors by regulating antitumor immunity.

PKCλ/ι inhibition activates an ULK2-mediated interferon response to repress tumorigenesis.

The interferon (IFN) pathway is critical for cytotoxic T cell activation, which is central to tumor immunosurveillance and successful immunotherapy. We demonstrate here that PKCλ/ι inactivation results in the hyper-stimulation of the IFN cascade and the enhanced recruitment of CD8+ T cells that impaired the growth of intestinal tumors. PKCλ/ι directly phosphorylates and represses the activity of ULK2, promoting its degradation through an endosomal microautophagy-driven ubiquitin-dependent mechanism. Loss of PKCλ/ι results in increased levels of enzymatically active ULK2, which, by direct phosphorylation, activates TBK1 to foster the activation of the STING-mediated IFN response. PKCλ/ι inactivation also triggers autophagy, which prevents STING degradation by chaperone-mediated autophagy. Thus, PKCλ/ι is a hub regulating the IFN pathway and three autophagic mechanisms that serve to maintain its homeostatic control. Importantly, single-cell multiplex imaging and bioinformatics analysis demonstrated that low PKCλ/ι levels correlate with enhanced IFN signaling and good prognosis in colorectal cancer patients.

Sterile inflammation via TRPM8 RNA-dependent TLR3-NF-kB/IRF3 activation promotes antitumor immunity in prostate cancer.

Inflammation is a common condition of prostate tissue, whose impact on carcinogenesis is highly debated. Microbial colonization is a well-documented cause of a small percentage of prostatitis cases, but it remains unclear what underlies the majority of sterile inflammation reported. Here, androgen- independent fluctuations of PSA expression in prostate cells have lead us to identify a prominent function of the Transient Receptor Potential Cation Channel Subfamily M Member 8 (TRPM8) gene in sterile inflammation. Prostate cells secret TRPM8 RNA into extracellular vesicles (EVs), which primes TLR3/NF-kB-mediated inflammatory signaling after EV endocytosis by epithelial cancer cells. Furthermore, prostate cancer xenografts expressing a translation-defective form of TRPM8 RNA contain less collagen type I in the extracellular matrix, significantly more infiltrating NK cells, and larger necrotic areas as compared to control xenografts. These findings imply sustained, androgen-independent expression of TRPM8 constitutes as a promoter of anticancer innate immunity, which may constitute a clinically relevant condition affecting prostate cancer prognosis.

S6K-STING interaction regulates cytosolic DNA-mediated activation of the transcription factor IRF3.

Cytosolic DNA-mediated activation of the transcription factor IRF3 is a key event in host antiviral responses. Here we found that infection with DNA viruses induced interaction of the metabolic checkpoint kinase mTOR downstream effector and kinase S6K1 and the signaling adaptor STING in a manner dependent on the DNA sensor cGAS. We further demonstrated that the kinase domain, but not the kinase function, of S6K1 was required for the S6K1-STING interaction and that the TBK1 critically promoted this process. The formation of a tripartite S6K1-STING-TBK1 complex was necessary for the activation of IRF3, and disruption of this signaling axis impaired the early-phase expression of IRF3 target genes and the induction of T cell responses and mucosal antiviral immunity. Thus, our results have uncovered a fundamental regulatory mechanism for the activation of IRF3 in the cytosolic DNA pathway.

The tumor suppressor PTEN has a critical role in antiviral innate immunity.

The gene encoding PTEN is one of the most frequently mutated tumor suppressor-encoding genes in human cancer. While PTEN's function in tumor suppression is well established, its relationship to anti-microbial immunity remains unknown. Here we found a pivotal role for PTEN in the induction of type I interferon, the hallmark of antiviral innate immunity, that was independent of the pathway of the kinases PI(3)K and Akt. PTEN controlled the import of IRF3, a master transcription factor responsible for IFN-ß production, into the nucleus. We further identified a PTEN-controlled negative phosphorylation site at Ser97 of IRF3 and found that release from this negative regulation via the phosphatase activity of PTEN was essential for the activation of IRF3 and its import into the nucleus. Our study identifies crosstalk between PTEN and IRF3 in tumor suppression and innate immunity.

Glutamylation of the DNA sensor cGAS regulates its binding and synthase activity in antiviral immunity.

Cyclic GMP-AMP synthase (cGAS) senses cytosolic DNA during viral infection and catalyzes synthesis of the dinucleotide cGAMP, which activates the adaptor STING to initiate antiviral responses. Here we found that deficiency in the carboxypeptidase CCP5 or CCP6 led to susceptibility to DNA viruses. CCP5 and CCP6 were required for activation of the transcription factor IRF3 and interferons. Polyglutamylation of cGAS by the enzyme TTLL6 impeded its DNA-binding ability, whereas TTLL4-mediated monoglutamylation of cGAS blocked its synthase activity. Conversely, CCP6 removed the polyglutamylation of cGAS, whereas CCP5 hydrolyzed the monoglutamylation of cGAS, which together led to the activation of cGAS. Therefore, glutamylation and deglutamylation of cGAS tightly modulate immune responses to infection with DNA viruses.

E3 ubiquitin ligase RNF128 promotes innate antiviral immunity through K63-linked ubiquitination of TBK1.

TBK1 is essential for interferon-ß (IFN-ß) production and innate antiviral immunity. Here we identified the T cell anergy-related E3 ubiquitin ligase RNF128 as a positive regulator of TBK1 activation. RNF128 directly interacted with TBK1 through its protease-associated (PA) domain and catalyzed the K63-linked polyubiquitination of TBK1, which led to TBK1 activation, IRF3 activation and IFN-ß production. Deficiency of RNF128 expression attenuated IRF3 activation, IFN-ß production and innate antiviral immune responses to RNA and DNA viruses, in vitro and in vivo. Our study identified RNF128 as an E3 ligase for K63-linked ubiquitination and activation of TBK1 and delineated a previously unrecognized function for RNF128.