IFN Regulatory Factor Research Articles

IFN regulatory factor-8 expression in macrophages governs an antimetastatic program.

High macrophage infiltration in cancer is associated with reduced survival in animal models and in patients. This reflects a shift in the macrophage response from a tumor-suppressive to tumor-supportive program governed by transcriptional events regulated by the inflammatory milieu. Although several transcription factors are known to drive a prometastatic program, those that govern an antimetastatic program are less understood. IFN regulatory factor-8 (IRF8) is integral for macrophage responses against infections. Using a genetic loss-of-function approach, we tested the hypothesis that IRF8 expression in macrophages governs their capacity to inhibit metastasis. We found that: (a) metastasis was significantly increased in mice with IRF8-deficient macrophages; (b) IRF8-deficient macrophages displayed a program enriched for genes associated with metastasis; and (c) lower IRF8 expression correlated with reduced survival in human breast and lung cancer, as well as melanoma, with high or low macrophage infiltration. Thus, a macrophagehiIRF8hi signature was more favorable than a macrophagehiIRF8lo signature. The same held true for a macrophageloIRF8hi vs. a macrophageloIRF8lo signature. These data suggest that incorporating IRF8 expression levels within a broader macrophage signature or profile strengthens prognostic merit. Overall, to our knowledge, our findings reveal a previously unrecognized role for IRF8 in macrophage biology to control metastasis or predict outcome.

IRF5 Is Required for Bacterial Clearance in Human M1-Polarized Macrophages, and IRF5 Immune-Mediated Disease Risk Variants Modulate This Outcome.

Common IFN regulatory factor 5 (IRF5) variants associated with multiple immune-mediated diseases are a major determinant of interindividual variability in pattern recognition receptor (PRR)-induced cytokines in macrophages. PRR-initiated pathways also contribute to bacterial clearance, and dysregulation of bacterial clearance can contribute to immune-mediated diseases. However, the role of IRF5 in macrophage-mediated bacterial clearance is not well defined. Furthermore, it is unclear if macrophages from individuals who are carriers of low IRF5-expressing genetic variants associated with protection for immune-mediated diseases might be at a disadvantage in bacterial clearance. We found that IRF5 was required for optimal bacterial clearance in PRR-stimulated, M1-differentiated human macrophages. Mechanisms regulated by IRF5 included inducing reactive oxygen species through p40phox, p47phox and p67phox, NOS2, and autophagy through ATG5. Complementing these pathways in IRF5-deficient M1 macrophages restored bacterial clearance. Further, these antimicrobial pathways required the activation of IRF5-dependent MAPK, NF-κB, and Akt2 pathways. Importantly, relative to high IRF5-expressing rs2004640/rs2280714 TT/TT immune-mediated disease risk-carrier human macrophages, M1-differentiated GG/CC carrier macrophages demonstrated less reactive oxygen species, NOS2, and autophagy pathway induction and, consequently, reduced bacterial clearance. Increasing IRF5 expression to the rs2004640/rs2280714 TT/TT levels restored these antimicrobial pathways. We define mechanisms wherein common IRF5 genetic variants modulate bacterial clearance, thereby highlighting that immune-mediated disease risk IRF5 carriers might be relatively protected from microbial-associated diseases.

Leishmania donovani Exploits Macrophage Heme Oxygenase-1 To Neutralize Oxidative Burst and TLR Signaling-Dependent Host Defense.

Suppression of host oxidative burst is essential for survival of the intracellular parasite Leishmania donovani Screening of macrophage antioxidant enzymes during infection revealed marked upregulation of the heme-degrading enzyme, heme oxygenase-1 (HO-1). Moreover, HO-1-silenced RAW macrophages depicted increased superoxide production and decreased parasite survival. HO-1 induction decreased cellular heme content, thereby inhibiting the heme-dependent maturation of gp91phox, a catalytic component of major reactive oxygen species-producing enzyme NAD(P)H oxidase. Decreased gp91phox expression resulted in reduced stability of p22phox, another component of the catalytic center of NAD(P)H oxidase. Replenishing infected cells with exogenous heme reversed these effects and restored NAD(P)H oxidase activity. Persistent HO-1 expression at late hour of infection prompted us to investigate its effect on other host defense parameters, and inhibition study revealed a reciprocal relationship of HO-1 with host proinflammatory responses. Among all the HO-1-mediated heme degradation products (CO, Fe, and biliverdin), only CO documented potent anti-inflammatory effects. Quenching of CO during infection increased the production of disease-resolving cytokines IL-12 and TNF-α. Coimmunoprecipitation experiments revealed that CO inhibited the interaction of TLR4 with MyD88 and TIR domain-containing adapter-inducing IFN-β, thereby dampening the activation of NF-κB and IFN regulatory factor 3-mediated production of proinflammatory cytokines. Administration of HO-1 inhibitor tin protoporphyrin IX dichloride in infected BALB/c mice led to a decrease in liver and spleen parasite burden along with increased production of IL-12 and TNF-α. These results suggest that HO-1 on one hand inhibits reactive oxygen species generation and on the other hand downregulates host favorable cytokine responses, thereby facilitating intramacrophage parasite survival.

IRF and STAT Transcription Factors - From Basic Biology to Roles in Infection, Protective Immunity, and Primary Immunodeficiencies.

The induction and action of type I interferon (IFN) is of fundamental importance in human immune defenses toward microbial pathogens, particularly viruses. Basic discoveries within the molecular and cellular signaling pathways regulating type I IFN induction and downstream actions have shown the essential role of the IFN regulatory factor (IRF) and the signal transducer and activator of transcription (STAT) families, respectively. However, the exact biological and immunological functions of these factors have been most clearly revealed through the study of inborn errors of immunity and the resultant infectious phenotypes in humans. The spectrum of human inborn errors of immunity caused by mutations in IRFs and STATs has proven very diverse. These diseases encompass herpes simplex encephalitis (HSE) and severe influenza in IRF3- and IRF7/IRF9 deficiency, respectively. They also include Mendelian susceptibility to mycobacterial infection (MSMD) in STAT1 deficiency, through disseminated measles infection associated with STAT2 deficiency, and finally staphylococcal abscesses and chronic mucocutaneous candidiasis (CMC) classically described with Hyper-IgE syndrome (HIES) in the case of STAT3 deficiency. More recently, increasing focus has been on aspects of autoimmunity and autoinflammation playing an important part in many primary immunodeficiency diseases (PID)s, as exemplified by STAT1 gain-of-function causing CMC and autoimmune thyroiditis, as well as a recently described autoinflammatory syndrome with hypogammaglobulinemia and lymphoproliferation as a result of STAT3 gain-of-function. Here I review the infectious, inflammatory, and autoimmune disorders arising from mutations in IRF and STAT transcription factors in humans, highlightning the underlying molecular mechanisms and immunopathogenesis as well as the clinical/therapeutic perspectives of these new insights.

The Molecular Basis of Viral Inhibition of IRF- and STAT-Dependent Immune Responses.

The antiviral innate immunity is the first line of host defense against virus infections. In mammalian cells, viral infections initiate the expression of interferons (IFNs) in the host that in turn activate an antiviral defense program to restrict viral replications by induction of IFN stimulated genes (ISGs), which are largely regulated by the IFN-regulatory factor (IRF) family and signal transducer and activator of transcription (STAT) family transcription factors. The mechanisms of action of IRFs and STATs involve several post-translational modifications, complex formation, and nuclear translocation of these transcription factors. However, many viruses, including human immunodeficiency virus (HIV), Zika virus (ZIKV), and herpes simplex virus (HSV), have evolved strategies to evade host defense, including alteration in IRF and STAT post-translational modifications, disturbing the formation and nuclear translocation of the transcription complexes as well as proteolysis/degradation of IRFs and STATs. In this review, we discuss and summarize the molecular mechanisms by which how viral components may target IRFs and STATs to antagonize the establishment of antiviral host defense. The underlying host-viral interactions determine the outcome of viral infection. Gaining mechanistic insight into these processes will be crucial in understanding how viral replication can be more effectively controlled and in developing approaches to improve virus infection outcomes.

Zebrafish NDRG1a Negatively Regulates IFN Induction by Promoting the Degradation of IRF7.

Viral infection activates the transcription factor IFN regulatory factor 7 (IRF7), which plays a critical role in the induction of IFNs and innate antiviral immune response. How virus-induced IFN signaling is controlled in fish is not fully understood. In this study, we demonstrate that N-myc downstream-regulated gene 1a (NDRG1a) in zebrafish plays a role as a negative regulator for virus-triggered IFN induction. First, the activation of the IFN promoter stimulated by the polyinosinic-polycytidylic acid or spring viremia of carp virus was decreased by the overexpression of NDRG1a. Second, NDRG1a interacted with IRF7 and blocked the IFN transcription activated by IRF7. Furthermore, NDRG1a was phosphorylated by TANK-binding kinase 1 (TBK1) and promoted the K48-linked ubiquitination and degradation of IRF7. Finally, the overexpression of NDRG1a blunted the transcription of several IFN-stimulated genes, resulting in the host cells becoming susceptible to spring viremia of carp virus infection. Our findings suggest that fish NDRG1a negatively regulates the cellular antiviral response by targeting IRF7 for ubiquitination and degradation, providing insights into the novel role of NDRG1a on the innate antiviral immune response in fish.

TLR3 Activation of Hepatic Stellate Cell Line Suppresses HBV Replication in HepG2 Cells.

There is limited information about the role of hepatic stellate cells (HSCs) in the liver innate immunity against hepatitis B virus (HBV) infection. We thus examined whether hepatic stellate cell line (LX-2) can be immunologically activated and produce antiviral factors that inhibit HBV replication in HepG2 cells. We found that LX-2 cells expressed the functional Toll-like receptor 3 (TLR3), activation of which by PolyI:C resulted in the selective induction of interferon-β (IFN-β) and IFN-λs, the phosphorylation of IFN regulatory factor 3 (IRF3) and IRF7. When HepG2 cells were treated with supernatant (SN) from PolyI:C-activated LX-2 cells, HBV replication was significantly inhibited. IFN-β and IFN-λ appeared to contribute to LX-2 SN-mediated HBV inhibition, as the antibodies to IFN-β and IFN-λ receptors could largely block the LX-2 SN action. Mechanistically, LX-2 SN treatment of the HepG2 cells induced a number of antiviral IFN-stimulated genes (ISGs: ISG20, ISG54, ISG56, OAS-1, Trim22, and Trim25) and facilitated the phosphorylation of STATs. These observations support further studies on the role of HSCs in the liver innate immunity against HBV infection.

Interferons Transcriptionally Up-Regulate MLKL Expression in Cancer Cells

Interferons (IFNs) are key players in the tumor immune response and act by inducing the expression of IFN-stimulated genes (ISGs). Here, we identify the mixed-lineage kinase domain-like pseudokinase (MLKL) as an ISG in various cancer cell lines. Both type I and type II IFNs increase the expression of MLKL indicating that MLKL up-regulation is a general feature of IFN signaling. IFNγ up-regulates mRNA as well as protein levels of MLKL demonstrating that IFNγ transcriptionally regulates MLKL. This notion is further supported by Actinomycin D chase experiments showing that IFNγ-stimulated up-regulation of MLKL is prevented in the presence of the transcriptional inhibitor Actinomycin D. Also, knockdown of the transcription factor IFN-regulatory factor 1 (IRF1) and signal transducer and activator of transcription (STAT) 1 as well as knockout of IRF1 significantly attenuate IFNγ-mediated induction of MLKL mRNA levels. Up-regulation of MLKL by IFNγ provides a valuable tool to sensitize cells towards necroptotic cell death and to overcome apoptosis resistance of cancer cells.

FKBP8 inhibits virus-induced RLR-VISA signaling.

The mitochondrial antiviral signal protein mitochondrial antiviral signaling protein, also known as virus-induced signaling adaptor (VISA), plays a key role in regulating host innate immune signaling pathways. This study identifies FK506 binding protein 8 (FKBP8) as a candidate interacting protein of VISA through the yeast two-hybrid technique. The interaction of FKBP8 with VISA, retinoic acid inducible protein 1 (RIG-I), and IFN regulatory factor 3 (IRF3) was confirmed during viral infection in mammalian cells by coimmunoprecipitation. Overexpression of FKBP8 using a eukaryotic expression plasmid significantly attenuated Sendai virus-induced activation of the promoter interferons β (IFN-β), and transcription factors nuclear factor κ-light chain enhancer of activated B cells (NF-κB) and IFN-stimulated response element (ISRE). Overexpression of FKBP8 also decreased dimer-IRF3 activity, but enhanced virus replication. Conversely, knockdown of FKBP8 expression by RNA interference showed opposite effects. Further studies indicated that FKBP8 acts as a negative interacting partner to regulate RLR-VISA signaling by acting on VISA and TANK binding kinase 1 (TBK1). Additionally, FKBP8 played a negative role on virus-induced signaling by inhibiting the formation of TBK1-IRF3 and VISA-TRAF3 complexes. Notably, FKBP8 also promoted the degradation of TBK1, RIG-I, and TRAF3 resulting from FKBP8 reinforced Sendai virus-induced endogenous polyubiquitination of RIG-I, TBK1, and TNF receptor-associated factor 3 (TRAF3). Therefore, a novel function of FKBP8 in innate immunity antiviral signaling regulation was revealed in this study.

BPIFA1 regulates lung neutrophil recruitment and interferon signaling during acute inflammation

Bpifa1 (BPI fold-containing group A member 1) is an airway host-protective protein with immunomodulatory properties that binds to LPS and is regulated by infectious and inflammatory signals. Differential expression of Bpifa1 has been widely reported in lung disease, yet the biological significance of this observation is unclear. We sought to understand the role of Bpifa1 fluctuations in modulating lung inflammation. We treated wild-type (WT) and Bpifa1-/- mice with intranasal LPS and performed immunological and transcriptomic analyses of lung tissue to determine the immune effects of Bpifa1 deficiency. We show that neutrophil (polymorphonuclear cells, PMNs) lung recruitment and transmigration to the airways in response to LPS is impaired in Bpifa1-/- mice. Transcriptomic analysis revealed a signature of 379 genes that differentiated Bpifa1-/- from WT mice. During acute lung inflammation, the most downregulated genes in Bpifa1-/- mice were Cxcl9 and Cxcl10. Bpifa1-/- mice had lower bronchoalveolar lavage concentrations of C-X-C motif chemokine ligand 10 (Cxcl10) and Cxcl9, interferon-inducible PMN chemokines. This was consistent with lower expression of IFNγ, IFNλ, downstream IFN-stimulated genes, and IFN-regulatory factors, which are important for the innate immune response. Administration of Cxcl10 before LPS treatment restored the inflammatory response in Bpifa1-/- mice. Our results identify a novel role for Bpifa1 in the regulation of Cxcl10-mediated PMN recruitment to the lungs via IFNγ and -λ signaling during acute inflammation.

Differential and Overlapping Immune Programs Regulated by IRF3 and IRF5 in Plasmacytoid Dendritic Cells.

We examined the signaling pathways and cell type-specific responses of IFN regulatory factor (IRF) 5, an immune-regulatory transcription factor. We show that the protein kinases IKKα, IKKβ, IKKε, and TANK-binding kinase 1 each confer IRF5 phosphorylation/dimerization, thus extending the family of IRF5 activator kinases. Among primary human immune cell subsets, we found that IRF5 is most abundant in plasmacytoid dendritic cells (pDCs). Flow cytometric cell imaging revealed that IRF5 is specifically activated by endosomal TLR signaling. Comparative analyses revealed that IRF3 is activated in pDCs uniquely through RIG-I-like receptor (RLR) signaling. Transcriptomic analyses of pDCs show that the partitioning of TLR7/IRF5 and RLR/IRF3 pathways confers differential gene expression and immune cytokine production in pDCs, linking IRF5 with immune regulatory and proinflammatory gene expression. Thus, TLR7/IRF5 and RLR-IRF3 partitioning serves to polarize pDC response outcome. Strategies to differentially engage IRF signaling pathways should be considered in the design of immunotherapeutic approaches to modulate or polarize the immune response for specific outcome.

Increase of viral hemorrhagic septicemia virus growth by knockout of IRF9 gene in Epithelioma papulosum cyprini cells

Viral hemorrhagic septicemia virus (VHSV) has been a notorious pathogen in freshwater and marine fish. Due to the lack of effective treatment measures against VHSV disease, the development of prophylactic vaccines has been required, and methods that can produce high-titered viruses would be advantageous in producing cost-effective vaccines. Type I interferon (IFN) responses are the key elements of vertebrates' antiviral activities, and IFN-stimulated gene factor 3 (ISGF3) complex formed through type I IFNs up-regulates the expression of IFN-stimulated genes (ISGs). IFN regulatory factor 9 (IRF9) is a key component of ISGF3, so the inhibition of IRF9 would compromise host's type I IFN responses, which would weaken host antiviral activity. In this study, to increase the replication of VHSV, we generated IRF9 knockout Epithelioma papulosum cyprini (EPC) cells using a CRISPR/Cas9 vector that contains an EPC cell's U6 promoter-driven guide RNA cassette (targeting IRF9 gene) and a Cas9 expressing cassette. In the clones of IRF9 knockout EPC cells, there were no increase in ISG15 gene by poly I:C, and in Mx1 gene by both poly I:C and VHSV. Interestingly, although the increased folds were conspicuously lower than control EPC cells, the expression of ISG 15 gene in all the IRF9 knockout clones was significantly increased by VHSV infection. Control EPC cells pre-treated with poly I:C did not show any CPE when infected with VHSV, however, IRF9 knockout EPC cells showed CPE by VHSV infection in spite of being pretreated with poly I:C. The replication of VHSV in IRF9 knockout EPC cells was significantly faster and higher than that in control EPC cells indicating that the IRF9 knockout-mediated decrease of type I IFN responses allowed VHSV to replicate efficiently. Considering an economical aspect for the production of fish vaccines, the present IRF9 knockout EPC cells can be used to get higher-titered VHSV.

Production of IL-35 by Bregs is mediated through binding of BATF-IRF-4-IRF-8 complex to il12a and ebi3 promoter elements.

IL-10 and IL-35 suppress excessive immune responses and therapeutic strategies are being developed to increase their levels in autoimmune diseases. In this study, we sought to identify major cell types that produce both cytokines in-vivo and to characterize mechanisms that regulate their production. Experimental autoimmune uveitis (EAU) is a CNS autoimmune disease that serves as model of human uveitis. We induced EAU in C57BL/6J mice and investigated whether T cells, B lymphocytes, or myeloid cells are the major producers of IL-10 or IL-35 in blood, lymph nodes (LNs), spleen, and at the site of ocular inflammation, the neuroretina. Analysis of these tissues identified B cells as the major producers of IL-10 and IL-35 in-vivo. Compared to regulatory T cells (Tregs), IL-10- or IL-35-producing regulatory B cells (Bregs) are substantially expanded in blood, LNs, spleen, and retina of mice with EAU. We performed EMSA and chromatin immunoprecipitation (ChIP) assays on activated B cells stimulated with IL-35 or TLR agonists. We found that BATF, IFN regulatory factor (IRF)-4, and IRF-8 transcription factors were recruited and bound to AP1-IRF-composite elements (AICEs) of il12a, ebi3, and/or il10 loci, suggesting their involvement in regulating IL-10 and IL-35 transcriptional programs of B cells. Showing that B cells are major source of IL-10 and IL-35 in-vivo and identifying transcription factors that contribute to IL-10 and IL-35 expression in the activated B-cell, suggest that the BATF/IRF-4/IRF-8 axis can be exploited therapeutically to regulate physiological levels of IL-10/IL-35-Bregs and that adoptive transfer of autologous Bregs might be an effective therapy for autoimmune and neurodegenerative diseases.

Variation in Genome-Wide NF-κB RELA Binding Sites upon Microbial Stimuli and Identification of a Virus Response Profile.

NF-κB transcription factors are master regulators of the innate immune response. Activated downstream of pathogen recognition receptors, they regulate the expression of genes to help fight infections as well as recruit the adaptive immune system. NF-κB responds to a wide variety of signals, but the processes by which stimulus specificity is attained remain unclear. In this article, we characterized the response of one NF-κB member, RELA, to four stimuli mimicking infection in human nasopharyngeal epithelial cells. Comparing genome-wide RELA binding, we observed stimulus-specific sites, although most sites overlapped across stimuli. Specifically, the response to poly I:C (mimicking viral dsRNA and signaling through TLR3) induced a distinct RELA profile, binding in the vicinity of antiviral genes and correlating with corresponding gene expression. This group of binding sites was also enriched in IFN regulatory factor motifs and showed overlapping with IFN regulatory factor binding sites. A novel NF-κB target, OASL, was further validated and showed TLR3-specific activation. This work showed that some RELA DNA binding sites varied in activation response following different stimulations and that interaction with more specialized factors could help achieve this stimulus-specific activity. Our data provide a genomic view of regulated host response to different pathogen stimuli.

Rhabdovirus-Inducible MicroRNA-210 Modulates Antiviral Innate Immune Response via Targeting STING/MITA in Fish.

Viral infection induces type I IFN production, which plays critical roles in orchestrating the antiviral defense by inducing direct antiviral activities. To establish a persistent infection, viruses have evolved numerous strategies to specifically interfere with IFN production or its downstream mediators, thereby evading the immune responses. MicroRNAs (miRNAs) are a family of small noncoding RNAs that posttranscriptionally regulate the expressions of specific target genes. Although accumulating evidence demonstrates that miRNAs play vital roles in regulating viral infection, miRNAs that target intracellular sensors and adaptors of innate immunity have not been fully uncovered. In this paper, we identify fish miR-210 as a robust regulator involved in regulating virus-host interactions. We found that rhabdovirus significantly upregulated the expression of fish miR-210. Inducible miR-210 modulates virus-triggered type I IFN and inflammatory cytokine production by targeting stimulator of IFN genes (STING), thereby promoting viral replication. Furthermore, we demonstrated that miR-210 regulates innate immune response through NF-κB, IFN regulatory factor 3, and JAK/STAT signaling pathways. The collective findings indicate that inducible miR-210 plays a regulatory role in virus-host interactions through STING-mediated singling pathway by targeting STING.

Tick-Borne Encephalitis Virus Nonstructural Protein NS5 Induces RANTES Expression Dependent on the RNA-Dependent RNA Polymerase Activity.

Tick-borne encephalitis virus (TBEV) is one of the flaviviruses that targets the CNS and causes encephalitis in humans. The mechanism of TBEV that causes CNS destruction remains unclear. It has been reported that RANTES-mediated migration of human blood monocytes and T lymphocytes is specifically induced in the brain of mice infected with TBEV, which causes ensuing neuroinflammation and may contribute to brain destruction. However, the viral components responsible for RANTES induction and the underlying mechanisms remain to be fully addressed. In this study, we demonstrate that the NS5, but not other viral proteins of TBEV, induces RANTES production in human glioblastoma cell lines and primary astrocytes. TBEV NS5 appears to activate the IFN regulatory factor 3 (IRF-3) signaling pathway in a manner dependent on RIG-I/MDA5, which leads to the nuclear translocation of IRF-3 to bind with RANTES promoter. Further studies reveal that the activity of RNA-dependent RNA polymerase (RdRP) but not the RNA cap methyltransferase is critical for TBEV NS5-induced RANTES expression, and this is likely due to RdRP-mediated synthesis of dsRNA. Additional data indicate that the residues at K359, D361, and D664 of TBEV NS5 are critical for RdRP activity and RANTES induction. Of note, NS5s from other flaviviruses, including Japanese encephalitis virus, West Nile virus, Zika virus, and dengue virus, can also induce RANTES expression, suggesting the significance of NS5-induced RANTES expression in flavivirus pathogenesis. Our findings provide a foundation for further understanding how flaviviruses cause neuroinflammation and a potential viral target for intervention.

Abstract 1932: Discovery of ENPP1 inhibitors as agonists of STING pathway

Abstract The discovery of STING (Stimulator of interferon genes) pathway and the second messenger 2′3′-cGAMP that activate it opened up several new possibilities for the development of anti-cancer and anti-pathogen therapeutics. STING is a pattern-recognition receptor anchored on endoplasmic reticulum and has pivotal roles in surveillance and innate immune response. Binding of 2′3′-cGAMP with STING induces conformational changes, which subsequently leads to perinuclear migration of STING and recruitment of TANK-binding kinase 1 (TBK1) and IFN regulatory factor 3 (IRF3). TBK1 phosphorylates IRF3, which then translocates to the nucleus and drives the transcription of innate immune genes, including IFN-β. Ectonucleotide pyrophosphatase/phosphodiesterase 1 (ENPP1) is a dominant hydrolyzing enzyme of 2′3′-cGAMP. In ENPP-/- cells, 2′3′-cGAMP is more active in stimulating STING activation, and this is predicted to be due to increased half-life. Similarly, hydrolysis resistant 2′3′-cGAMP analogs are reported to be potent STING agonists. We hypothesized that inhibition of ENPP1 activity with an orally available small molecule would be a novel approach to activate STING pathway when compared with the cyclic dinucleotides (CDNs) due to their parenteral route of administration. We have utilized computational methods successfully discovered small molecule candidates that showed a strong binding affinity towards ENPP1. With the help of in vitro enzymatic inhibition and direct binding assays we have identified few lead candidates with an IC50 of <100 nM. Cellular assays further showed target specific inhibition of ENPP1 activity and subsequent activation of STING pathway. Additionally, our lead series of compounds showed no effect on other members of the ENPP family, a kinome panel, and several epigenetic regulators, suggesting the specificity towards ENPP1. In summary, we have developed small molecule inhibitors of ENPP1 and one of our lead candidate's is currently under investigation for ADME-Tox, PK and efficacy. The results from these studies will be presented. Citation Format: Sunil Sharma, Alexis Weston, Trason Thode, Eric Gomez, Mohan Kaadige, Hariprasad Vankayalapti. Discovery of ENPP1 inhibitors as agonists of STING pathway [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2018; 2018 Apr 14-18; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2018;78(13 Suppl):Abstract nr 1932.

Ctenopharyngodon idella TBK1 activates innate immune response via IRF7

In mammals, IFN regulatory factor (IRF) 7 is a central regulator of IFN-α expression in response to variable pathogenic infections. There are several pathogenic sensors involved in monitoring pathogen intrusion in mammals. These sensors trigger IRF7-mediated responses through different pathways. TANK-binding kinase 1 (TBK1) is a critical mediator of IRF7 activation upon pathogen infection. In fish, there are many reports on TBK1, IRF3 and IRF7, especially on TBK1-IRF3 signaling pathway. However, it is not very clear how TBK1-IRF7 works in innate immune signaling pathway. In this study, we explored how TBK1 up-regulates IFN, ISG expression, and how TBK1 initiates innate immune response through IRF7 in fish under lipopolysaccharides (LPS) stimulation. After stimulation with LPS, grass carp IRF3 and IRF7 transcriptions were up-regulated, indicating they participate in TLR-mediated antiviral signaling pathway. It is interesting that the response time of grass carp IRF3 to LPS was earlier than that of IRF7. In addition, IRF7 rather than IRF3 acted as a stronger positive regulator of IFN and ISG transcription in Ctenopharyngodon idella kidney cells (CIKs). It is suggested the potential function differentiation between IRF3 and IRF7 upon LPS infection in fish. Dual luciferase assays also showed that overexpression of grass carp IRF7 and TBK1 up-regulated the transcription level of IFN and PKR. However, knockdown of IRF7 inhibits ISG expression, suggesting that grass carp TBK1 regulates the transcription via IRF7. Co-immunoprecipitation and GST pull-down assays proved the binding of grass carp IRF7 to TBK1. Furthermore, grass carp TBK1 can promote the nuclear translocation of IRF7. The results indicated that grass carp TBK1 can bind directly to and activate IRF7.

SOCS1 and SOCS3 Target IRF7 Degradation To Suppress TLR7-Mediated Type I IFN Production of Human Plasmacytoid Dendritic Cells

Type I IFN production of plasmacytoid dendritic cells (pDCs) triggered by TLR-signaling is an essential part of antiviral responses and autoimmune reactions. Although it was well-documented that members of the cytokine signaling (SOCS) family regulate TLR-signaling, the mechanism of how SOCS proteins regulate TLR7-mediated type I IFN production has not been elucidated yet. In this article, we show that TLR7 activation in human pDCs induced the expression of SOCS1 and SOCS3. SOCS1 and SOCS3 strongly suppressed TLR7-mediated type I IFN production. Furthermore, we demonstrated that SOCS1- and SOCS3-bound IFN regulatory factor 7, a pivotal transcription factor of the TLR7 pathway, through the SH2 domain to promote its proteasomal degradation by lysine 48-linked polyubiquitination. Together, our results demonstrate that SOCS1/3-mediated degradation of IFN regulatory factor 7 directly regulates TLR7 signaling and type I IFN production in pDCs. This mechanism might be targeted by therapeutic approaches to either enhance type I IFN production in antiviral treatment or decrease type I IFN production in the treatment of autoimmune diseases.

Hu Antigen R Regulates Antiviral Innate Immune Responses through the Stabilization of mRNA for Polo-like Kinase 2.

Retinoic acid-inducible gene I (RIG-I)-like receptors (RLRs), RIG-I, and melanoma differentiation-associated gene 5 (MDA5) play a critical role in inducing antiviral innate immune responses by activating IFN regulatory factor 3 (IRF3) and NF-κB, which regulates the transcription of type I IFN and inflammatory cytokines. Antiviral innate immune responses are also regulated by posttranscriptional and translational mechanisms. In this study, we identified an RNA-binding protein HuR as a regulator for RLR signaling. Overexpression of HuR, but not of other Hu members, increased IFN-β promoter activity. HuR-deficient macrophage cells exhibited decreased Ifnb1 expression after RLR stimulation, whereas they showed normal induction after stimulation with bacterial LPS or immunostimulatory DNA. Moreover, HuR-deficient cells displayed impaired nuclear translocation of IRF3 after RLR stimulation. In HuR-deficient cells, the mRNA expression of Polo-like kinase (PLK) 2 was markedly reduced. We found that HuR bound to the 3' untranslated region of Plk2 mRNA and increased its stabilization. PLK2-deficient cells also showed reduced IRF3 nuclear translocation and Ifnb mRNA expression during RLR signaling. Together, these findings suggest that HuR bolsters RLR-mediated IRF3 nuclear translocation by controlling the stability of Plk2 mRNA.