Inflammatory cell and cytokine cascade activation is present in humans with alcoholic liver disease as well as in animal models of alcohol-induced liver damage. Gut-derived lipopolysaccharide (LPS), a ligand of the Toll-like receptor 4 (TLR4), plays a central role in triggering and maintaining activation of Kupffer cells in alcoholic hepatitis. In this mini-review, we describe molecular mechanisms that lead to increased inflammatory cell activation by alcohol and LPS and discuss the mechanism for activation in alcohol-exposed macrophages. In alcohol-induced liver disease we discuss the role of MyD88-independent but IRF3-mediated TLR4 signaling in alcohol-related liver inflammation and liver damage.

Varicella-zoster virus (VZV) is an alphaherpesvirus that is restricted to humans. VZV infection of differentiated cells within the host and establishment of latency likely require evasion of innate immunity and limited secretion of antiviral cytokines. Since interferons (IFNs) severely limit VZV replication, we examined the ability of VZV to modulate the induction of the type I IFN response in primary human embryonic lung fibroblasts (HELF). IFN-beta production was not detected, and transcription of two interferon response factor 3 (IRF3)-dependent interferon-stimulated genes (ISGs), ISG54 and ISG56, in response to poly(I:C) stimulation was downregulated in VZV-infected HELF. Inhibition of IRF3 function did not require VZV replication; the viral immediate-early protein 62 (IE62) alone was sufficient to produce this effect. IE62 blocked TBK1-mediated IFN-beta secretion and IRF3 function, as shown in an IFN-stimulated response element (ISRE)-luciferase reporter assay. However, IRF3 function was preserved if constitutively active IRF3 (IRF3-5D) was expressed in VZV-infected or IE62-transfected cells, indicating that VZV interferes with IRF3 phosphorylation. IE62-mediated inhibition was mapped to blocking phosphorylation of at least three serine residues on IRF3. However, IE62 binding to TBK1 or IRF3 was not detected and IE62 did not perturb TBK1-IRF3 complex formation. IE62-mediated inhibition of IRF3 function was maintained even if IE62 transactivator activity was disrupted. Thus, IE62 has two critical but discrete roles following VZV entry: to induce expression of VZV genes and to disarm the IFN-dependent antiviral defense through a novel mechanism that prevents IRF3 phosphorylation.

Modified vaccinia virus Ankara (MVA) is an attenuated poxvirus that has been engineered as a vaccine against infectious agents and cancers. Our goal is to understand how MVA modulates innate immunity in dendritic cells (DCs), which can provide insights to vaccine design. In this study, using murine bone marrow-derived dendritic cells, we assessed type I interferon (IFN) gene induction and protein secretion in response to MVA infection. We report that MVA infection elicits the production of type I IFN in murine conventional dendritic cells (cDCs), but not in plasmacytoid dendritic cells (pDCs). Transcription factors IRF3 (IFN regulatory factor 3) and IRF7, and the positive feedback loop mediated by IFNAR1 (IFN alpha/beta receptor 1), are required for the induction. MVA induction of type I IFN is fully dependent on STING (stimulator of IFN genes) and the newly discovered cytosolic DNA sensor cGAS (cyclic guanosine monophosphate-adenosine monophosphate synthase). MVA infection of cDCs triggers phosphorylation of TBK1 (Tank-binding kinase 1) and IRF3, which is abolished in the absence of cGAS and STING. Furthermore, intravenous delivery of MVA induces type I IFN in wild-type mice, but not in mice lacking STING or IRF3. Treatment of cDCs with inhibitors of endosomal and lysosomal acidification or the lysosomal enzyme Cathepsin B attenuated MVA-induced type I IFN production, indicating that lysosomal enzymatic processing of virions is important for MVA sensing. Taken together, our results demonstrate a critical role of the cGAS/STING-mediated cytosolic DNA-sensing pathway for type I IFN induction in cDCs by MVA. We present evidence that vaccinia virulence factors E3 and N1 inhibit the activation of IRF3 and the induction of IFNB gene in MVA-infected cDCs.

A novel SARS-related coronavirus (SARS-CoV-2) has recently emerged as a serious pathogen that causes high morbidity and substantial mortality. However, the mechanisms by which SARS-CoV-2 evades host immunity remain poorly understood. Here, we identified SARS-CoV-2 membrane glycoprotein M as a negative regulator of the innate immune response. We found that the M protein interacted with the central adaptor protein MAVS in the innate immune response pathways. This interaction impaired MAVS aggregation and its recruitment of downstream TRAF3, TBK1, and IRF3, leading to attenuation of the innate antiviral response. Our findings reveal a mechanism by which SARS-CoV-2 evades the innate immune response and suggest that the M protein of SARS-CoV-2 is a potential target for the development of SARS-CoV-2 interventions.

Keywords: Innate immunity; MAVS; Membrane glycoprotein M; SARS-CoV-2; Type I interferon.

Mesenchymal tumor subpopulations secrete pro-tumorigenic cytokines and promote treatment resistance1-4. This phenomenon has been implicated in chemorefractory small cell lung cancer and resistance to targeted therapies5-8, but remains incompletely defined. Here, we identify a subclass of endogenous retroviruses (ERVs) that engages innate immune signaling in these cells. Stimulated 3 prime antisense retroviral coding sequences (SPARCS) are oriented inversely in 3' untranslated regions of specific genes enriched for regulation by STAT1 and EZH2. Derepression of these loci results in double-stranded RNA generation following IFN-γ exposure due to bi-directional transcription from the STAT1-activated gene promoter and the 5' long terminal repeat of the antisense ERV. Engagement of MAVS and STING activates downstream TBK1, IRF3, and STAT1 signaling, sustaining a positive feedback loop. SPARCS induction in human tumors is tightly associated with major histocompatibility complex class 1 expression, mesenchymal markers, and downregulation of chromatin modifying enzymes, including EZH2. Analysis of cell lines with high inducible SPARCS expression reveals strong association with an AXL/MET-positive mesenchymal cell state. While SPARCS-high tumors are immune infiltrated, they also exhibit multiple features of an immune-suppressed microenviroment. Together, these data unveil a subclass of ERVs whose derepression triggers pathologic innate immune signaling in cancer, with important implications for cancer immunotherapy.

Porcine reproductive and respiratory syndrome (PRRS) is an emerged disease of swine characterized by negligible response of type I IFNs and viral persistence. We show that the PRRSV non-structural protein 1 (Nsp1) is the viral component responsible for modulation of IFN response. Nsp1 blocked dsRNA-induced IRF3 and IFN promoter activities. Nsp1 did not block phosphorylation and nuclear translocation of IRF3 but inhibited IRF3 association with CREB-binding protein (CBP) in the nucleus. While IRF3 was stable, CBP was degraded, and CBP degradation was proteasome-dependent, suggesting that CBP degradation is not due to the protease activity of Nsp1 but an intermediary is involved. Our data suggest that the Nsp1-mediated CBP degradation inhibits the recruitment of CBP for enhanceosome assembly, leading to the block of IFN response. CBP degradation is a novel strategy for viral evasion from the host response, and Nsp1 may form a new class of viral antagonists for IFN modulation.

RNAi (RNA interference) has become a powerful tool to determine gene function. Different methods of expressing the short ds (double-stranded) RNA intermediates required for interference in mammalian systems have been developed, including the introduction of si (short interfering) RNAs by direct transfection or driven from transfected plasmids or lentiviral vectors encoding sh (short hairpin) RNAs. Although RNAi relies upon a high degree of specificity, recent findings suggest that off-target non-specific effects can be encountered. We found that transfection of siRNAs can results in an interferon-mediated activation of the JAK/STAT (Janus kinase/signal transducer and activator of transcription) pathway and global up-regulation of interferon-stimulated genes. This effect is mediated in part by the dsRNA-dependent protein kinase PKR, as this kinase is activated by the 21 bp siRNA, and is required in response to the siRNAs. However, the transcription factor IRF3 (interferon-regulatory factor 3) is also activated by siRNA as a primary response, resulting in the stimulation of genes independent of an interferon response. In cells deficient in IRF3, this response is blunted, but can be restored by re-introduction of IRF3. Thus siRNAs induce complex signalling responses in target cells, leading to effects beyond the selective silencing of specific genes.

In mammals, IFN regulatory factor (IRF) 3 is a critical player in modulating transcription of type I IFN and IFN-stimulated genes (ISGs). In this study, we describe the roles of crucian carp (Carassius auratus L.) IRF3 in activating fish IFN and ISGs. Fish IRF3 exhibits a large sequence divergence from mammalian orthologs. Whereas mammalian IRF3 is constitutively expressed, fish IRF3 protein is significantly upregulated by IFN, poly-IC, and other stimuli known as IFN inducers in mammals. The IFN-inducible property of fish IRF3 is consistent with the comparative analysis of 5' flanking regulatory region of vertebrate IRF3 genes, which reveals the presence of typical IFN-stimulated response elements in fish and amphibians, but an absence in tetrapods. Furthermore, either IFN or poly-IC induces phosphorylation and cytoplasmic-to-nuclear translocation of IRF3, which seems essential for its function in that phosphomimic active IRF3 exhibits stronger transactivation than wild type IRF3. Finally, overexpression of fish IRF3 activates production of IFN that in turn triggers ISG transcription through Stat1 pathway, whereas transfection of dominant negative mutant IRF3-DN abrogates poly-IC induction of ISGs, probably owing to blockade of IFN production. Therefore, regulation of IFN response by vertebrate IRF3 is another ancient trait. These data provide evidence of the evolving function of vertebrate IRF3 on regulating IFN response.

Pattern recognition receptors (PRRs) in innate immune cells play a pivotal role in the first line of host defense system. PRRs recognize pathogen-associated molecular patterns (PAMPs) or danger-associated molecular patterns (DAMPs) to initiate and regulate innate and adaptive immune responses. PRRs include Toll-like receptors (TLRs), RIG-I-like receptors (RLRs) and NOD-like receptors (NLRs), which have their own features in ligand recognition and cellular location. Activated PRRs deliver signals to adaptor molecules (MyD88, TRIF, MAL/TIRAP, TRAM, IPS-1) which act as important messengers to activate downstream kinases (IKK complex, MAPKs, TBK1, RIP-1) and transcription factors (NF-κB, AP-1, IRF3), which produce effecter molecules including cytokines, chemokines, inflammatory enzymes, and type I interferones. Since excessive PRR activation is closely linked to the development of chronic inflammatory diseases, the role of intrinsic and extrinsic regulators in the prevention of over- or unnecessary activation of PRRs has been widely studied. Intracellular regulators include MyD88s, SOCS1, TOLLIP, A20, and CYLD. Extrinsic regulators have also been identified with their molecular targets in PRR signaling pathways. TLR dimerization has been suggested as an inhibitory target for small molecules such as curcumin, cinnamaldehyde, and sulforaphane. TBK1 kinase can be a target for certain flavonoids such as EGCG, luteolin, quercetin, chrysin, and eriodictyol to regulate TRIF-dependent TLR pathways. This review focuses on the features of PRR signaling pathways and the therapeutic targets of intrinsic and extrinsic regulators in order to provide beneficial strategies for controlling the activity of PRRs and the related inflammatory diseases and immune disorders.

Virus infection leads to the activation of transcription factor IRF3 and subsequent production of type I inteferons, which induce the transcription of various antiviral genes called interferon stimulated genes (ISGs) to eliminate viral infection. IRF3 activation requires phosphorylation, dimerization and nuclear translocation. However, the mechanisms for the termination of IRF3 activation in nucleus are elusive. Here we report the identification of TRIM26 to negatively regulate IFN-β production and antiviral response by targeting nuclear IRF3. TRIM26 bound to IRF3 and promoted its K48-linked polyubiquitination and degradation in nucleus. TRIM26 degraded WT IRF3 and the constitutive active mutant IRF3 5D, but not the phosphorylation deficient mutant IRF3 5A. Furthermore, IRF3 mutant in the Nuclear Localization Signal (NLS), which could not move into nucleus, was not degraded by TRIM26. Importantly, virus infection promoted TRIM26 nuclear translocation, which was required for IRF3 degradation. As a consequence, TRIM26 attenuated IFN-β promoter activation and IFN-β production downstream of TLR3/4, RLR and DNA sensing pathways. TRIM26 transgenic mice showed much less IRF3 activation and IFN-β production, while increased virus replication. Our findings delineate a novel mechanism for the termination of IRF3 activation in nucleus through TRIM26-mediated IRF3 ubiquitination and degradation.

The innate immune system pattern recognition receptors (PRR) are the first line of host defenses recognizing the various pathogen- or danger-associated molecular patterns and eliciting defenses by regulating the production of pro-inflammatory cytokines such as IL-1β, IL-18 or interferon β (IFN-β). NOD-like receptors (NLRs) and AIM2-like receptors (ALRs) are cytoplasmic inflammasome sensors of foreign molecules, including DNA. IFI16, a sequence-independent nuclear innate sensor ALR, recognizes episomal dsDNA genomes of herpes viruses such as KSHV, EBV, and HSV-1 in the infected cell nuclei, forms an inflammasome complex with ASC and procaspase1, and relocates into the cytoplasm leading into Caspase-1 and IL-1β generation. IFI16 also induces IFN-β during HSV-1 infection via the cytoplasmic STING-TBK1-IRF3 pathway. Thus far, whether IFI16 recognizes foreign DNA directly or utilizes other host protein(s) is unknown. Here, we demonstrate that BRCA1, a DNA damage repair sensor and transcription regulator, is in complex with IFI16 in the host cell nucleus, and their association increases in the presence of nuclear viral genomes during de novo KSHV, EBV and HSV-1 infection, and in latent KSHV or EBV infection, but not by DNA damage responses (DDR) induced by bleomycin and vaccinia virus cytoplasmic dsDNA. BRCA1 is a constituent of the triggered IFI16-inflammasome and is translocated into the cytoplasm after genome recognition along with the IFI16-inflammasome. The absence of BRCA1 abrogated IFI16-viral genome association, inflammasome assembly, IFI16 cytoplasmic localization, and Caspase-1 and IL-1β production. The absence of BRCA1 also abolished the cytoplasmic IFI16-STING interaction, downstream IRF3 phosphorylation, nuclear translocation of pIRF3 and IFN-β production during de novo KSHV and HSV-1 infection. These findings highlight that BRCA1 plays a hitherto unidentified innate immunomodulatory role by facilitating nuclear foreign DNA sensing by IFI16, subsequent assembly and cytoplasmic distribution of IFI16-inflammasomes leading into IL-1β formation and the induction of IFN-β via cytoplasmic signaling through IFI16-STING, TBK1 and IRF3.

Phosphorylation of the transcription factor interferon regulatory factor 3 (IRF3) is essential for the induction of promoters which contain the interferon-stimulated response element (ISRE). IRF3 can be activated by Toll-like receptor 3 (TLR3) in response to the double-stranded RNA mimic poly(I-C) and by TLR4 in response to lipopolysaccharide (LPS). Here we have analyzed the effect of the glucocorticoid dexamethasone on this response. Dexamethasone inhibited the induction of the ISRE-dependent gene RANTES (regulated on activation normal T cell expressed and secreted) in both U373-CD14 cells and human peripheral blood mononuclear cells and also an ISRE luciferase construct, activated by either TLR3 or TLR4. It also inhibited increased phosphorylation of IRF3 in its N terminus in response to LPS and in its C terminus on Ser-396 in response to either poly(I-C) or LPS. Several dexamethasone-induced phosphatases were tested for possible involvement in these effects; MKP1 did not appear to be involved, although MKP2 and MKP5 both partially inhibited induction of the ISRE, pointing to their possible involvement in the effect of dexamethasone. Importantly, we found that dexamethasone could inhibit TBK1 kinase activity and TBK1 phosphorylation on Ser-172, both of which are required for IRF3 phosphorylation downstream of TLR3 and TLR4 stimulation. Our study, therefore, demonstrates that TBK1 is a target for dexamethasone, common to both TLR3 and TLR4 signaling.

In the CNS, microglia are the primary targets of HIV infection. In this study, we investigated the effect of activation of the innate antiviral receptors TLR3 and TLR4 on HIV infection of primary human microglia, as well as microglial cell signaling and gene expression. Ligands for both TLR3 and TLR4 potently inhibited HIV replication in microglia through a pathway requiring IRF3. Surprisingly, a remarkably similar pattern of cell signaling and gene expression was observed in TLR3- and TLR4-activated microglia, suggesting a relatively minor role for MyD88 following TLR4 activation in these cells. HIV did not activate IRF3 but rather decreased IRF3 protein, indicating that HIV does not activate TLR3 or RIG-like helicases in microglia. Taken together, these results indicate that activation of TLR3 or TLR4 will elicit antiviral immunity, in addition to inducing proinflammatory responses. We suggest that a balanced expression between inflammatory and innate immune genes might be achieved by IRF3 over-expression.

The Interferon regulatory factors (IRFs) are a family of transcription factors that play pivotal roles in many aspects of the immune response, including immune cell development and differentiation and regulating responses to pathogens. Three family members, IRF3, IRF5, and IRF7, are critical to production of type I interferons downstream of pathogen recognition receptors that detect viral RNA and DNA. A fourth family member, IRF9, regulates interferon-driven gene expression. In addition, IRF4, IRF8, and IRF5 regulate myeloid cell development and phenotype, thus playing important roles in regulating inflammatory responses. Thus, understanding how their levels and activity is regulated is of critical importance given that perturbations in either can result in dysregulated immune responses and potential autoimmune disease. This review will focus the role of IRF family members in regulating type I IFN production and responses and myeloid cell development or differentiation, with particular emphasis on how regulation of their levels and activity by ubiquitination and microRNAs may impact autoimmune disease.

CpG-rich oligodeoxynucleotides activate the immune system, leading to innate and acquired immune responses. The immune-stimulatory effects of CpG-rich oligodeoxynucleotides are being exploited as a therapeutic approach. Here we show that at high doses, CpG-rich oligodeoxynucleotides promote an opposite, tolerogenic response in mouse plasmacytoid dendritic cells in vivo and in a human in vitro model. Unveiling a previously undescribed role for TRIF and TRAF6 proteins in Toll-like receptor 9 (TLR9) signalling, we demonstrate that physical association of TLR9, TRIF and TRAF6 leads to activation of noncanonical NF-κB signalling and the induction of IRF3- and TGF-β-dependent immune-suppressive tryptophan catabolism. In vivo, the TLR9-TRIF circuit--but not MyD88 signalling--was required for CpG protection against allergic inflammation. Our findings may be relevant to an increased understanding of the complexity of Toll-like receptor signalling and optimal exploitation of CpG-rich oligodeoxynucleotides as immune modulators.

Interferon regulatory factor 3 (IRF3) is known to participate in the transcriptional induction of interferon (IFN) alpha and IFNbeta genes, as well as of a number of interferon-stimulated genes (ISGs), as a result of viral infection. In the present study we demonstrate the activation of IRF3 followed by ISG induction after exposure of cells to the bacterial cell wall component lipopolysaccharide. Engagement of Toll-like receptors by lipopolysaccharide triggered the nuclear translocation of IRF3, followed by its DNA binding and the subsequent induction of several interferon-regulated genes. Transcriptional activation of ISGs occurred in a protein synthesis independent manner, but was sensitive to inhibition of the stress-activated protein kinase, p38. The activation of IRF3 by viral particles or bacterial membrane components suggests that this signaling pathway might contribute to the evolutionary conserved innate immune response.

TLRs signal the presence of microbial patterns and activate transcription factors. In TLR3 and TLR4, the adapter Toll-IL-1R homology domain-containing adapter molecule (TICAM-1) (also called Toll/IL-1R domain-containing adapter inducing IFN-beta (TRIF)) mediates IFN regulatory factor 3 (IRF3) phosphorylation followed by IFN-beta production. The regulatory subunit TNFR-associated factor family member-associated NF-kappaB activator (TANK) couples with the kinase complex IkappaB kinase-related kinase epsilon/NF-kappaB-activating kinase (NAK) (TANK-binding kinase 1 (TBK1)) that involveTICAM-1-dependent IFN-beta induction. There are several TANK-homologous proteins. We tested whether TICAM-1 binds and coprecipitates with TANK or its family proteins. The results are: 1) the TANK family protein NAK-associated protein 1 (NAP1), but not TANK, coprecipitates withTICAM-1; 2) NAP1 overexpression markedly enhances TBK1-mediated IFN-beta promoter activation; 3) a dominant-negative form, NAP (158-270), suppresses IRF3 activation in response to poly(I:C) or LPS; 4) RNA interference targeting of the NAP1 message results in a failure of poly(I:C)-mediated IRF3 polymerization and IFN-beta production. Thus, NAP1 is the kinase subunit responsible for TLR3/4-mediated IFN-beta induction in the TICAM-1 pathway.

Detection of microbes by TLRs on the plasma membrane leads to the induction of proinflammatory cytokines such as TNF-α, via activation of NF-κB. Alternatively, activation of endosomal TLRs leads to the induction of type I IFNs via IFN regulatory factors (IRFs). TLR4 signaling from the plasma membrane to NF-κB via the Toll/IL-1R (TIR) adaptor protein MyD88 requires the TIR sorting adaptor Mal, whereas endosomal TLR4 signaling to IRF3 via the TIR domain-containing adaptor-inducing IFN-β (TRIF) requires the TRIF-related adaptor molecule (TRAM). Similar to TLR4 homodimers, TLR2 heterodimers can also induce both proinflammatory cytokines and type I IFNs. TLR2 plasma membrane signaling to NF-κB is known to require MyD88 and Mal, whereas endosomal IRF activation by TLR2 requires MyD88. However, it was unclear whether TLR2 requires a sorting adaptor for endosomal signaling, like TLR4 does. In this study, we show that TLR2-dependent IRF7 activation at the endosome is both Mal- and TRAM-dependent, and that TRAM is required for the TLR2-dependent movement of MyD88 to endosomes following ligand engagement. TRAM interacted with both TLR2 and MyD88, suggesting that TRAM can act as a bridging adapter between these two molecules. Furthermore, infection of macrophages lacking TRAM with herpes viruses or the bacterium Staphylococcus aureus led to impaired induction of type I IFN, indicating a role for TRAM in TLR2-dependent responses to human pathogens. Our work reveals that TRAM acts as a sorting adaptor not only for TLR4, but also for TLR2, to facilitate signaling to IRF7 at the endosome, which explains how TLR2 is capable of causing type I IFN induction.

OBJECTIVE

AMPK activates SIRT1 in liver and skeletal muscle. Impaired circadian function is associated with development of obesity. SIRT1 regulates circadian function and is suppressed in white adipose tissue (WAT) of obese patients. We examined the potential role of AMPK and SIRT1 in regulation of circadian components in WAT of obese db/db mice and in mice fed a high-fat diet (HFD), and investigated whether metformin-mediated activation of AMPK opposed any deleterious changes in the WAT clock mechanism.

METHODS

db/+ and db/db mice were administered metformin (250 mg/kg/day; 7 days). Separately, mice were fed HFD for 16-weeks. 3T3-L1 adipocytes were incubated with metformin, EX527 or FK866, inhibitors of SIRT1 and NAMPT, respectively. Gene and protein expression were measured by qRT-PCR and immunoblotting.

RESULTS

AMPK activity, NAMPT expression and SIRT1 expression were decreased in WAT of db/db and HFD mice, in association with suppressed expression of the core circadian components CLOCK and BMAL1. Expression of Pparγ and the adipogenic repressors Irf3 and Irf4 were also suppressed. Metformin increased AMPK activity in WAT of db/db mice and in metformin-treated adipocytes, with increased NAMPT, SIRT1 and circadian component expression. Metformin-mediated induction of Clock mRNA in adipocytes was blocked by inhibition of NAMPT and SIRT1.

CONCLUSIONS

Decreased AMPK-SIRT1 signalling in db/db and HFD mice impacts WAT circadian function causing dysregulated lipid regulation, favouring an obese phenotype. Metformin mediates a phenotypic shift away from lipid accretion through AMPK-NAMPT-SIRT1 mediated changes in clock components, supporting chronotherapeutic treatment approaches for obesity.

Chronic inflammation is an important component that contributes to many age-related neurodegenerative diseases, including macular degeneration. Here, we report a role for toll-like receptor 3 (TLR3) in cone-rod dystrophy (CORD) of mice lacking ATP-binding cassette transporter 4 (ABCA4) and retinol dehydrogenase 8 (RDH8), proteins critical for all-trans-retinal clearance in the retina. Increased expression of toll-like receptor-signaling elements and inflammatory changes were observed in Rdh8(-/-)Abca4(-/-) eyes by RNA expression analysis. Unlike 3-month-old Rdh8(-/-)Abca4(-/-) mice that developed CORD, 6-month-old Tlr3(-/-)Rdh8(-/-)Abca4(-/-) mice did not evidence an abnormal retinal phenotype. Light-induced retinal degeneration in Tlr3(-/-)Rdh8(-/-)Abca4(-/-) mice was milder than that in Rdh8(-/-)Abca4(-/-) mice, and a 2-fold increased TLR3 expression was detected in light-illuminated retinas of Rdh8(-/-)Abca4(-/-) mice compared with nonilluminated retinas. Poly(I-C), a TLR3 ligand, caused caspase-8-independent cellular apoptosis. Whereas poly(I-C) induced retinal cell death in Rdh8(-/-)Abca4(-/-) and WT mice both in vivo and ex vivo, this was not seen in mice lacking Tlr3. Far fewer invasive macrophage/microglial cells in the subretinal space and weaker activation of Muller glial cells were exhibited by Tlr3(-/-)Rdh8(-/-) Abca4(-/-) mice compared with Rdh8(-/-)Abca4(-/-) mice at 3 and 6 months of age, indicating that loss of TLR3 inhibits local inflammation in the retina. Both poly(I-C) and endogenous products emanating from dying/dead retinal cells induced NF-κB and IRF3 activation. These findings demonstrate that endogenous products from degenerating retina stimulate TLR3 that causes cellular apoptosis and retinal inflammation and that loss of TLR3 protects mice from CORD.

The balance between the innate immunity of the host and the ability of a pathogen to evade it strongly influences pathogenesis and virulence. The two nonstructural (NS) proteins, NS1 and NS2, of respiratory syncytial virus (RSV) are critically required for RSV virulence. Together, they strongly suppress the type I interferon (IFN)-mediated innate immunity of the host cells by degrading or inhibiting multiple cellular factors required for either IFN induction or response pathways, including RIG-I, IRF3, IRF7, TBK1 and STAT2. Here, we provide evidence for the existence of a large and heterogeneous degradative complex assembled by the NS proteins, which we named "NS-degradasome" (NSD). The NSD is roughly ∼300-750 kD in size, and its degradative activity was enhanced by the addition of purified mitochondria in vitro. Inside the cell, the majority of the NS proteins and the substrates of the NSD translocated to the mitochondria upon RSV infection. Genetic and pharmacological evidence shows that optimal suppression of innate immunity requires mitochondrial MAVS and mitochondrial motility. Together, we propose a novel paradigm in which the mitochondria, known to be important for the innate immune activation of the host, are also important for viral suppression of the innate immunity.

The pattern recognition receptor RIG-I is critical for Type-I interferon production. However, the global regulation of RIG-I signaling is only partially understood. Using a human genome-wide RNAi-screen, we identified 226 novel regulatory proteins of RIG-I mediated interferon-β production. Furthermore, the screen identified a metabolic pathway that synthesizes the inositol pyrophosphate 1-IP7 as a previously unrecognized positive regulator of interferon production. Detailed genetic and biochemical experiments demonstrated that the kinase activities of IPPK, PPIP5K1 and PPIP5K2 (which convert IP5 to1-IP7) were critical for both interferon induction, and the control of cellular infection by Sendai and influenza A viruses. Conversely, ectopically expressed inositol pyrophosphate-hydrolases DIPPs attenuated interferon transcription. Mechanistic experiments in intact cells revealed that the expression of IPPK, PPIP5K1 and PPIP5K2 was needed for the phosphorylation and activation of IRF3, a transcription factor for interferon. The addition of purified individual inositol pyrophosphates to a cell free reconstituted RIG-I signaling assay further identified 1-IP7 as an essential component required for IRF3 activation. The inositol pyrophosphate may act by β-phosphoryl transfer, since its action was not recapitulated by a synthetic phosphonoacetate analogue of 1-IP7. This study thus identified several novel regulators of RIG-I, and a new role for inositol pyrophosphates in augmenting innate immune responses to viral infection that may have therapeutic applications.

Viral infection activates interferon regulatory factor 3 (IRF3), a cofactor for the induction of interferon-stimulated genes (ISGs). The role of IRF3 in the activation of ISGs by human cytomegalovirus (HCMV) is controversial despite the fact that HCMV has consistently been shown to induce ISGs during infection of fibroblasts. To address the function of IRF3 in HCMV-mediated ISG induction, we monitored ISG expression and global gene expression in HCMV-infected cells in which IRF3 function had been depleted by small interfering RNA or blocked by dominant negative IRF3. A specific reduction of ISG induction was observed, whereas other transcripts were unaffected. We therefore conclude that IRF3 specifically regulates ISG induction during the initial phase of HCMV infection.

Immunotherapy is one of the key strategies for cancer treatment. The cGAS-cGAMP-STING-IRF3 pathway of cytosolic DNA sensing plays a pivotal role in antiviral defense. We report that the STING activator cGAMP possesses significant antitumor activity in mice by triggering the STING-dependent pathway directly. cGAMP enhances innate immune responses by inducing production of cytokines such as interferon-β, interferon-γ, and stimulating dendritic cells activation, which induces the cross-priming of CD8(+) T cells. The antitumor mechanism of cGAMP was verified by STING and IRF3, which were up-regulated upon cGAMP treatment. STING-deficiency dramatically reduced the antitumor effect of cGAMP. Furthermore, cGAMP improved the antitumor activity of 5-FU, and clearly reduced the toxicity of 5-FU. These results demonstrated that cGAMP is a novel antitumor agent and has potential applications in cancer immunotherapy.