Transcription Factor IRF3 Research Articles

Polarization of Macrophage and Lupus Nephritis

Lupus nephritis (LN) is one of the common and severe complications in systemic lupus erythematosus (SLE), and is a leading cause of morbidity and mortality for SLE patients. It is well known that LN is characterized by the inflammation mediated by immune response in the kidney tissues, during which macrophage (Mo) plays a vital role. According to the varied microenvironments in different stages of lupus nephritis, macrophages are divided into two categories, namely classically activated macrophages (M1) and alternatively activated macrophages (M2). Macrophages can undergo phenotypical/functional switch and play different functions depending on multiple signal pathways, including STAT transcription factors, epigenetic aspects, NF-κB pathways, IRF transcription factors and some interleukins, chemokines and its receptors. Due to the heterogeneity and plasticity, the polarization of macrophages may exert an influence on the outcome of lupus nephritis. Thus, targeting the polarization of macrophages properly may become a new therapeutic treatment for lupus nephritis. This review focuses on how polarization of macrophages regulates the pathogenesis of LN.

Abstract B031: Heat-inactivated modified vaccinia virus ankara induces type I IFN and antitumor immunity via the cytosolic DNA-sensing pathway

Abstract Type I interferon (IFN), well known for its antiviral activity, is a critical component of cancer immune surveillance through its direct actions on cancer cells as well as on tumor microenvironment. Advanced melanoma remains a therapeutic challenge despite recent progress in targeted therapy and immunotherapy. Novel approaches are needed to alter the tumor immune suppressive microenvironment and to facilitate the recognition of tumor antigens that leads to antitumor immunity. Poxviruses are cytosolic DNA viruses that have been investigated as oncolytic and immunotherapeutic agents. We recently reported that the highly attenuated modified vaccinia virus Ankara (MVA), a safe vaccine for smallpox, triggers type I IFN production in conventional dendritic cells (cDCs) via the cytosolic DNA sensor cGAS and its adaptor STING, and that it requires transcription factors IRF3 and IRF7. Here we show that infection of cDCs with heat-inactivated MVA leads to higher levels of IFN induction than MVA. This induction is also mediated by the cytosolic DNA-sensing pathway cGAS/STING/IRF3/IRF7. In addition, we found that intratumoral injection of Heat-MVA caused tumor eradication in a murine B16 melanoma model as well as the generation of adaptive anti-tumor immunity. Furthermore, Heat-MVA-induced anti-tumor therapy is less effective in STING, IRF7, or Batf3-deficient mice than in wild-type mice, indicating that both the innate immune-sensing pathway and CD8α+DCs are essential for Heat-MVA-based immunotherapy. Lastly, the combination of intratumoral delivery of Heat-MVA with systemic delivery of anti-CTLA-4, PD-1 or PD-L1 antibodies achieved enhanced efficacy in tumor eradication and survival than Heat-MVA alone. Our results have strong implications for the development of poxvirus-based cancer immunotherapeutics as well novel strategies to overcome resistance to immune checkpoint blockade therapy. Citation Format: Peihong Dai, Weiyi Wang, Cristian Serna-Tamayo, Dimitriy Zamarin, Stewart Shuman, Taha Merghoub, Jedd D. Wolchok, Liang Deng. Heat-inactivated modified vaccinia virus ankara induces type I IFN and antitumor immunity via the cytosolic DNA-sensing pathway. [abstract]. In: Proceedings of the CRI-CIMT-EATI-AACR Inaugural International Cancer Immunotherapy Conference: Translating Science into Survival; September 16-19, 2015; New York, NY. Philadelphia (PA): AACR; Cancer Immunol Res 2016;4(1 Suppl):Abstract nr B031.

Dynamics of REL, RELA and IRF1 transcription factor expression in U937 macrophages after dioxin exposure

Dynamics of REL, RELA and IRF1 transcription factor expression in U937 macrophages after dioxin exposure

Membrane Perturbation-Associated Ca2+ Signaling and Incoming Genome Sensing Are Required for the Host Response to Low-Level Enveloped Virus Particle Entry.

The type I interferon (IFN) response is an important aspect of innate antiviral defense, and the transcription factor IRF3 plays an important role in its induction. Membrane perturbation during fusion, a necessary step for enveloped virus particle entry, appears sufficient to induce transcription of a subset of IFN-stimulated genes (ISGs) in an IRF3-dependent, IFN-independent fashion. IRF3 is emerging as a central node in host cell stress responses, although it remains unclear how different forms of stress activate IRF3. Here, we investigated the minimum number of Sendai virus (SeV) and human cytomegalovirus (HCMV) particles required to activate IRF3 and trigger an antiviral response. We found that Ca(2+) signaling associated with membrane perturbation and recognition of incoming viral genomes by cytosolic nucleic acid receptors are required to activate IRF3 in response to fewer than 13 particles of SeV and 84 particles of HCMV per cell. Moreover, it appears that Ca(2+) signaling is important for activation of STING and IRF3 following HCMV particle entry, suggesting that Ca(2+) signaling sensitizes cells to recognize genomes within incoming virus particles. To our knowledge, this is the first evidence that cytosolic nucleic acid sensors recognize genomes within incoming virus particles prior to virus replication. These studies highlight the exquisite sensitivity of the cellular response to low-level stimuli and suggest that virus particle entry is sensed as a stress signal. The mechanism by which replicating viruses trigger IRF3 activation and type I IFN induction through the generation and accumulation of viral pathogen-associated molecular patterns has been well characterized. However, the mechanism by which enveloped virus particle entry mediates a stress response, leading to IRF3 activation and the IFN-independent response, remained elusive. Here, we find that Ca(2+) signaling associated with membrane perturbation appears to sensitize cells to recognize genomes within incoming virus particles. To our knowledge, this is the first study to show that cytosolic receptors recognize genomes within incoming virus particles prior to virus replication. These findings not only highlight the sensitivity of cellular responses to low-level virus particle stimulation, but provide important insights into how nonreplicating virus vectors or synthetic lipid-based carriers used as clinical delivery vehicles activate innate immune responses.

Roles for Transcription Factors Sp1, NF-κB, IRF3, and IRF7 in Expression of the Human IFNL4 Gene.

The expression of a biologically active human IFNλ4 depends on the presence of a frameshift deletion polymorphism within the first exon of the interferon lambda 4 (IFNL4) gene. In this report, we use the lung carcinoma-derived cell line, A549, which is genetically viable to express a functional IFNλ4, to address transcriptional requirements of the IFNL4 gene. We show that the GC-rich DNA-binding transcription factor (TF) specificity protein 1 (Sp1) is recruited to the IFNL4 promoter and has a role in induction of gene expression upon stimulation with viral RNA mimic poly(I:C). By using RNAi and overexpression strategies, we also show key roles in IFNL4 gene expression for the virus-inducible TFs, nuclear factor kappa-light-chain-enhancer of activated B cells (NF-κB), IFN regulatory factor 3 (IRF3), and IRF7. Interestingly, we also observe that overexpression of IFNλ4 influences IFNL4 promoter activity, which may further be dependent on the retinoic acid-inducible gene-I (RIG-I)-like receptor pathway. Together, our work for the first time reports on the functional characterization of the human IFNL4 promoter.

ACY-241, a Novel, HDAC6 Selective Inhibitor: Synergy with Immunomodulatory (IMiD®) Drugs in Multiple Myeloma (MM) Cells and Early Clinical Results (ACE-MM-200 Study)

Histone deacetylase (HDAC) enzymes are attractive therapeutic targets in oncology, but non-selective HDAC inhibitors have led to dose-limiting toxicities in patients, particularly in combination with other therapeutic agents. Ricolinostat (ACY-1215), a first-in-class orally available HDAC inhibitor that is 11-fold selective for HDAC6, synergizes in vitro and in vivo in models of MM and lymphoma with bortezomib (Santo, Blood, 2012; Amengual, Clin Cancer Res, 2015) or carfilzomib (Mishima, Br J Haematol, 2015; Dasmahapatra, Mol Cancer Ther, 2014). Furthermore, ricolinostat has demonstrated an excellent safety and tolerability profile in phase I trials as an oral liquid formulation (Raje, Haematologica, 2014, Suppl 1). We have now identified ACY-241 as a structurally related and orally available selective inhibitor of HDAC6 that is undergoing clinical evaluation in tablet form.In combination with ricolinostat, the immunomodulatory (IMiD®) class of drugs, including lenalidomide (Len) and pomalidomide (Pom), exhibit striking anti-myeloma properties in a variety of MM models (Quayle, AACR, 2014) and have demonstrated clinical activity in MM patients (Yee, ASH, 2014). In support of our ongoing development of ACY-241, we show here that combination with either Len or Pom leads to synergistic decrease in MM cell viability in vitro. Time course studies demonstrated cell cycle arrest followed by progressive induction of apoptosis after prolonged exposure to Len or Pom. Notably, the addition of ACY-241 to either Len or Pom resulted in synergistic increases in apoptosis of MM cells. At the molecular level, treatment with IMiDs reduced expression of the critical transcription factors MYC and IRF4, which was further reduced by combination treatment with ACY-241. Current studies are exploring the molecular mechanism underlying this effect, which may be a consequence of low level inhibition of HDAC1, 2, and 3 by ACY-241. Prolonged treatment with ACY-241 plus Pom was well tolerated in vivo with no evidence of toxicity, and the combination resulted in a significant extension of survival in a xenograft model of MM. Given the comparable tolerability profiles of ricolinostat and ACY-241 and the similar preclinical activity in combination with IMiDs, a clinical trial (NCT02400242) is currently evaluating ACY-241 in combination with Pom and low-dose dexamethasone in MM patients.Predicated upon the clinical experience with ricolinostat and the non-clinical pharmacokinetics of ACY-241, we designed an expedited first-in-human phase 1a/1b clinical trial of a single cycle of ACY-241 monotherapy followed by ACY-241 in combination with Pom and dexamethasone in MM patients. A merged monotherapy/combination trial design was chosen to grant patients access to combination therapy with an established regimen while enabling insight into the safety, pharmacokinetics, and pharmacodynamics of ACY-241 monotherapy. Patients with relapsed or relapsed-and-refractory MM previously treated with at least two cycles of Len and a proteasome inhibitor were eligible for this trial. The first patient was enrolled in June 2015. This patient tolerated monotherapy well and pharmacokinetics showed maximal plasma levels of ACY-241 in the micromolar range, consistent with predictions. An update on enrollment, pharmacokinetic and pharmacodynamic profiles as well as safety of monotherapy and combination therapy will be provided. DisclosuresNiesvizky:Celgene: Consultancy, Speakers Bureau. Richardson:Jazz Pharmaceuticals: Membership on an entity's Board of Directors or advisory committees, Research Funding; Novartis: Membership on an entity's Board of Directors or advisory committees; Millennium Takeda: Membership on an entity's Board of Directors or advisory committees; Gentium S.p.A.: Membership on an entity's Board of Directors or advisory committees, Research Funding; Celgene Corporation: Membership on an entity's Board of Directors or advisory committees. Gabrail:Onyx: Honoraria, Speakers Bureau; BI: Honoraria, Speakers Bureau; Janssen: Speakers Bureau; Sanofi: Honoraria, Speakers Bureau. Madan:Onyx: Speakers Bureau; Celgene: Speakers Bureau. Quayle:Acetylon Pharmaceuticals, Inc.: Employment, Equity Ownership. Almeciga-Pinto:Acetylon Pharmaceuticals, Inc: Employment. Jones:Acetylon Pharmaceuticals, Inc.: Employment, Equity Ownership. Houston:Acetylon Pharmaceuticals, Inc: Employment. Hayes:Acetylon Pharmaceuticals, Inc: Employment. Van Duzer:Acetylon Pharmaceuticals, Inc: Employment. Wheeler:Acetylon Pharmaceuticals, INC: Employment. Trede:Acetylon Pharmaceuticals, Inc: Employment. Raje:Acetylon: Research Funding; Celgene Corporation: Consultancy; BMS: Consultancy; Amgen: Consultancy; Millenium: Consultancy; AstraZeneca: Research Funding; Novartis: Consultancy; Onyx: Consultancy; Eli Lilly: Research Funding; Takeda: Consultancy.

Single Cell RNA seq for Analysis of Cell Fate Decisions

Single-cell RNA-Seq has the potential to become a dominant approach in probing diverse and complex developmental compartments. Its unbiased and comprehensive nature could enable developmental ordering of cellular and regulatory gene hierarchies without prior knowledge. To test general utility we performed single-cell RNA-seq of murine hematopoietic progenitors focusing on the myeloid developmental hierarchy. Using novel unsupervised clustering analysis, ICDS, we correctly ordered known hierarchical states as well as revealed rare intermediates. Regulatory state analysis suggested that the transcription factors Gfi1 and Irf8 function antagonistically to control homeostatic neutrophil and macrophage production, respectively. This prediction was validated by complementary genetic and genomic experiments in granulocyte-macrophage progenitors. Using knock-in reporters for Gfi1 and Irf8 and clonogenic analyses coupled with single-cell RNA-seq we distinguished regulatory states of bi-potential progenitors from their lineage specifying or committed progeny. Thus single-cell RNA-Seq is a powerful developmental tool to characterize hierarchical and rare cellular states along with the regulators that control their dynamics. DisclosuresNo relevant conflicts of interest to declare.

Regulation of bifurcating B cell trajectories by mutual antagonism between transcription factors IRF4 and IRF8

Upon recognition of antigen, B cells undertake a bifurcated response in which some cells rapidly differentiate into plasmablasts while others undergo affinity maturation in germinal centers (GCs). Here we identified a double-negative feedback loop between the transcription factors IRF4 and IRF8 that regulated the initial developmental bifurcation of activated B cells as well as the GC response. IRF8 dampened signaling via the B cell antigen receptor (BCR), facilitated antigen-specific interaction with helper T cells, and promoted antibody affinity maturation while antagonizing IRF4-driven differentiation of plasmablasts. Genomic analysis revealed concentration-dependent actions of IRF4 and IRF8 in regulating distinct gene-expression programs. Stochastic modeling suggested that the double-negative feedback was sufficient to initiate bifurcation of the B cell developmental trajectories.

Autoubiquitination of TRIM26 links TBK1 to NEMO in RLR-mediated innate antiviral immune response.

The transcription factors IRF3 and NF-κB are required for the expression of many genes involved in antiviral innate immune response, including type I interferons (IFNs) and proinflammatory cytokines. It is well established that TBK1 is an essential kinase engaged downstream of multiple pattern-recognition receptors (PRRs) to mediate IRF3 phosphorylation and activation, whereas the precise mechanisms of TBK1 activation have not been fully elucidated yet. Here, we identified tripartite motif 26 (TRIM26) as an important regulator for RNA virus-triggered innate immune response. Knockdown of TRIM26 impaired virus-triggered IRF3, NF-κB activation, IFN-β induction, and cellular antiviral response. TRIM26 was physically associated with TBK1 independent of viral infection. As an E3 ligase, TRIM26 underwent autoubiquitination upon viral infection. Ubiquitinated TRIM26 subsequently associated with NEMO, thus bridging TBK1-NEMO interaction, which is critical for the recruitment of TBK1 to the VISA signalsome and activation of TBK1. Our findings suggest that TRIM26 is an important regulator of innate immune responses against RNA viruses, which functions by bridging TBK1 to NEMO and mediating the activation of TBK1.

Respiratory DC Use IFITM3 to Avoid Direct Viral Infection and Safeguard Virus-Specific CD8+ T Cell Priming.

Respiratory dendritic cells (DC) play a pivotal role in the initiation of adaptive immune responses to influenza virus. To do this, respiratory DCs must ferry viral antigen from the lung to the draining lymph node without becoming infected and perishing en route. We show that respiratory DCs up-regulate the expression of the antiviral molecule, interferon-induced transmembrane protein 3 (IFITM3) in response to influenza virus infection, in a manner dependent on type I interferon signaling and the transcription factors IRF7 and IRF3. Failure of respiratory DCs to up-regulate IFITM3 following influenza virus infection resulted in impaired trafficking to the draining LN and consequently in impaired priming of an influenza-specific CD8+ T cell response. The impaired trafficking of IFITM3-deficient DC correlated with an increased susceptibility of these DC to influenza virus infection. This work shows that the expression of IFITM3 protects respiratory DCs from influenza virus infection, permitting migration from lung to LN and optimal priming of a virus specific T-cell response.

Prior infection with Type A Francisella tularensis antagonizes the pulmonary transcriptional response to an aerosolized Toll-like receptor 4 agonist.

BackgroundFrancisella infection attenuates immune cell infiltration and expression of selected pro-inflammatory cytokines in response to endogenous LPS, suggesting the bacteria is actively antagonizing at least some part of the response to Toll-like receptor 4 (TLR4) engagement. The ability of different Francisella strains to inhibit the ability of E. coli LPS to induce a pulmonary inflammatory response, as measured by gene expression profiling, was examined to define the scope of modulation and identify of inflammatory genes/pathways that are specifically antagonized by a virulent F. tularensis infection.ResultsPrior aerosol exposure to F. tularensis subsp. tularensis, but not the live attenuated strain (LVS) of F. tularensis subsp. holarctica or F. novicida, significantly antagonized the transcriptional response in the lungs of infected mice exposed to aerosolized E. coli LPS. The response to E. coli LPS was not completely inhibited, suggesting that the bacteria is targeting further downstream of the TLR4 molecule. Analysis of the promotors of LPS-responsive genes that were perturbed by Type A Francisella infection identified candidate transcription factors that were potentially modulated by the bacteria, including multiple members of the forkhead transcription factor family (FoxA1, Foxa2, FoxD1, Foxd3, Foxf2, FoxI1, Fox03, Foxq1), IRF1, CEBPA, and Mef2. The annotated functional roles of the affected genes suggested that virulent Francisella infection suppressed cellular processes including mRNA processing, antiviral responses, intracellular trafficking, and regulation of the actin cytoskeleton. Surprisingly, despite the broad overall suppression of LPS-induced genes by virulent Francisella, and contrary to what was anticipated from prior studies, Type A Francisella did not inhibit the expression of the majority of LPS-induced cytokines, nor the expression of many classic annotated inflammatory genes.ConclusionsCollectively, this analysis demonstrates clear differences in the ability of different Francisella strains to modulate TLR4 signaling and identifies genes/pathways that are specifically targeted by virulent Type A Francisella.Electronic supplementary materialThe online version of this article (doi:10.1186/s12864-015-2022-2) contains supplementary material, which is available to authorized users.

Interferon-Regulatory Factor 5-Dependent Signaling Restricts Orthobunyavirus Dissemination to the Central Nervous System.

Interferon (IFN)-regulatory factor 5 (IRF-5) is a transcription factor that induces inflammatory responses after engagement and signaling by pattern recognition receptors. To define the role of IRF-5 during bunyavirus infection, we evaluated Oropouche virus (OROV) and La Crosse virus (LACV) pathogenesis and immune responses in primary cells and in mice with gene deletions in Irf3, Irf5, and Irf7 or in Irf5 alone. Deletion of Irf3, Irf5, and Irf7 together resulted in uncontrolled viral replication in the liver and spleen, hypercytokinemia, extensive liver injury, and an early-death phenotype. Remarkably, deletion of Irf5 alone resulted in meningoencephalitis and death on a more protracted timeline, 1 to 2 weeks after initial OROV or LACV infection. The clinical signs in OROV-infected Irf5(-/-) mice were associated with abundant viral antigen and terminal deoxynucleotidyltransferase-mediated dUTP-biotin nick end labeling (TUNEL)-positive cells in several regions of the brain. Circulating dendritic cell (DC) subsets in Irf5(-/-) mice had higher levels of OROV RNA in vivo yet produced lower levels of type I IFN than wild-type (WT) cells. This result was supported by data obtained in vitro, since a deficiency of IRF-5 resulted in enhanced OROV infection and diminished type I IFN production in bone marrow-derived DCs. Collectively, these results indicate a key role for IRF-5 in modulating the host antiviral response in peripheral organs that controls bunyavirus neuroinvasion in mice. Oropouche virus (OROV) and La Crosse virus (LACV) are orthobunyaviruses that are transmitted by insects and cause meningitis and encephalitis in subsets of individuals in the Americas. Recently, we demonstrated that components of the type I interferon (IFN) induction pathway, particularly the regulatory transcription factors IRF-3 and IRF-7, have key protective roles during OROV infection. However, the lethality in Irf3(-/-) Irf7(-/-) (DKO) mice infected with OROV was not as rapid or complete as observed in Ifnar(-/-) mice, indicating that other transcriptional factors associated with an IFN response contribute to antiviral immunity against OROV. Here, we evaluated bunyavirus replication, tissue tropism, and cytokine production in primary cells and mice lacking IRF-5. We demonstrate an important role for IRF-5 in preventing neuroinvasion and the ensuing encephalitis caused by OROV and LACV.

Proteolysis of MDA5 and IPS-1 is not required for inhibition of the type I IFN response by poliovirus.

BackgroundThe type I interferon (IFN) response is a critical component of the innate immune response to infection by RNA viruses and is initiated via recognition of viral nucleic acids by RIG-like receptors (RLR). Engagement of these receptors in the cytoplasm initiates a signal transduction pathway leading to activation of the transcription factors NF-κB, ATF-2 and IRF-3 that coordinately upregulate transcription of type I IFN genes, such as that encoding IFN-β. In this study the impact of poliovirus infection on the type I interferon response has been examined.MethodsThe type I IFN response was assessed by measuring IFN-β mRNA levels using qRT-PCR and normalizing to levels of β-actin mRNA. The status of host factors involved in activation of the type I IFN response was examined by immunoblot, immunofluorescence microcopy and qRT-PCR.ResultsThe results show that poliovirus infection results in induction of very low levels of IFN-β mRNA despite clear activation of NF-κB and ATF-2. In contrast, analysis of IRF-3 revealed no transcriptional induction of an IRF-3-responsive promoter or homodimerization of IRF-3 indicating it is not activated in poliovirus-infected cells. Exposure of poliovirus-infected cells to poly(I:C) results in lower levels of IFN-β mRNA synthesis and IRF-3 activation compared to mock-infected cells. Analysis of MDA-5 and IPS-1 revealed that these components of the RLR pathway were largely intact at times when the type I IFN response was suppressed.ConclusionsCollectively, these results demonstrate that poliovirus infection actively suppresses the host type I interferon response by blocking activation of IRF-3 and suggests that this is not mediated by cleavage of MDA-5 or IPS-1.

S1P promotes breast cancer progression by angiogenesis and lymphangiogenesis.

S1P promotes breast cancer progression by angiogenesis and lymphangiogenesis.

DYRK2 Negatively Regulates Type I Interferon Induction by Promoting TBK1 Degradation via Ser527 Phosphorylation.

Viral infection activates the transcription factors NF-κB and IRF3, which contribute to the induction of type I interferons (IFNs) and cellular antiviral responses. Protein kinases play a critical role in various signaling pathways by phosphorylating their substrates. Here, we identified dual-specificity tyrosine-(Y)-phosphorylation-regulated kinase 2 (DYRK2) as a negative regulator of virus-triggered type I IFN induction. DYRK2 inhibited the virus-triggered induction of type I IFNs and promoted the K48-linked ubiquitination and degradation of TANK-binding kinase 1 (TBK1) in a kinase-activity-dependent manner. We further found that DYRK2 phosphorylated Ser527 of TBK1, which is essential for the recruitment of NLRP4 and for the E3 ubiquitin ligase DTX4 to degrade TBK1. These findings suggest that DYRK2 negatively regulates virus-triggered signaling by targeting TBK1 for phosphorylation and priming it for degradation, and these data provide new insights into the molecular mechanisms that dictate the cellular antiviral response.

Systemic IL‐1β production as a consequence of corneal HSV‐1 infection – contribution to the development of Herpes Simplex Keratitis

PurposeHerpes Simplex Virus type‐1 (HSV‐1) infection can result in keratitis, a sight‐threatening disease that is the leading cause of infectious corneal blindness in the western world. HSV‐1 can invade the cornea and remain latent after treatment in the trigeminal ganglia. The pathogenesis of HSK involves a complex interaction between cytokines, chemokines and growth factors, either brought in by inflammatory cells or produced locally. Avoidance of these innate anti‐viral responses can cause lifelong recurrent infection, which in turn can cause progressive corneal scarring, vascularisation, thinning and the need for corneal transplantation to recover vision, often with poor long‐term outcome. This study aimed to investigate peripheral cytokine production in HSK patients with active and inactive infection to identify potential therapeutic targets.MethodsPeripheral blood mononuclear cells and serum were prepared from whole blood taken from both healthy controls and HSK patients during active infection or following treatment. Protein expression levels were analysed by Western Blot. Cytokine levels were determined by multiplex ELISA.ResultsActive corneal HSV‐1 infection resulted in significantly elevated peripheral levels of the pro‐inflammatory cytokine IL‐1b in patients compared to healthy controls. IL‐1β levels remained significantly increased in these patients following treatment. Impaired production of pro‐inflammatory cytokines (including IL‐6, IL‐8 and TNF‐a) and anti‐viral factors (including IL‐12 and IFN‐g) was associated with significantly reduced expression of the transcription factors IRF3 and STAT1 in active patients compared to inactive patients.ConclusionsOur data suggest that dysregulated peripheral production of pro‐inflammatory cytokines and anti‐viral factors may have implications for HSV‐1 viral clearance and recurrent keratitis.

ID: 113: STING, regulated by RIG-I signaling, contributes to 5’ppp RNA restriction of HSV via sustaining IFN response

RIG-I is essential for recognizing RNA viruses with a 5’ triphosphate (ppp) signature in the cytoplasm. Upon viral RNA recognition, RIG-I recruits adaptor protein MAVS to trigger the activation of IRF3 and NF-kB transcription factors, leading to activation of antiviral response. STING has been identified as an RIG-I signaling cofactor and a critical adaptor protein required for cytosolic DNA and cyclic dinucleotides (CDNs) triggered immune responses. We have identified STING among a plethora of differentially expressed genes induced by the 5’ppp RNA; in the present study, we further detail the mechanism of STING regulation. Our data shows that virus infection induces STING expression at both the mRNA and protein levels. Furthermore STING induction is shown to be dependent on functional RIG-I signaling. STING induction by the RIG-I agonist 5’ppp RNA was recognized as a delayed event resulting from an autocrine/paracrine mechanism. Indeed, co-treatment with TNF α and IFN α has a synergistic effect on the regulation of STING expression. Depletion of STAT1 and RELA, either individually or combined, significantly diminished virus-induced STING protein expression compared with the cells. In A549 cells, the expression of interferon-response/inflammatory response genes was not affected in the absence of STING at early time points, but was significantly reduced at late time points. Furthermore, our results also unveiled an essential contribution of STING in the establishment of the 5’pppRNA induced antiviral responses during HSV1 infection. Taken together, these observations demonstrate that the STING is induced via RIG-I signaling and up-regulated STING is essential for 5’ppp RNA mediated HSV restriction.

ID: 82: The Innate Immune Sensor, LGP2, Interferes with TRAF Proteins to Prevent NFkB Activation

The detection of RNA virus infections triggers the production type I interferon (IFN), and is a major component of the powerful antiviral system that directly interferes with virus replication and contributes to both innate and adaptive immune responses. Virus replication intermediates such as double stranded RNA (dsRNA) or RNAs with distinct non-self modifications are recognized in the cytoplasm by pattern recognition receptors including RIG-I, MDA5, and LGP2. These innate sensors recognize non-self RNAs via their C-terminal domains, and RIG-I and MDA5 use N-terminal CARDs to induce the activation of the downstream signaling scaffold, MAVS. MAVS coordinates the assembly of signaling machinery including TRAFs and IKK-family kinases that activate IRF3 and NFkB transcription factors. Available evidence suggests LGP2 acts as a concentration dependent switch to regulate RLR antiviral signaling. For positive actions, LGP2 uses its unique catalytic activity to enhance MDA5 RNA recognition, dsRNA filament assembly, and enable more efficient activation of MAVS and downstream signal transduction. The mechanism of LGP2 negative regulation have been investigated, and results indicate that LGP2-mediated inhibition occurs downstream of RIG-I, MDA5, and MAVS. LGP2 inhibition does not require ATPase activity or RNA binding. Instead, results indicate that LGP2 targets TRAF signaling proteins, disengaging their ubiquitin ligase activity, and potently preventing NFkB activation in MAVS dependent and independent systems. Identification of TRAF proteins as inhibitory targets not only resolves the dual roles of LGP2 in antiviral signaling regulation, but also expands the potential impact of LGP2 negative regulation beyond innate antiviral signaling.

ID: 183: Overcoming cell-to-cell heterogeneity reveals novel dynamic properties of IRF-3 and IFN-β

The type-I Interferon (IFN) response is an early innate immunity component and is antagonised by factors of a broad range of pathogenic viruses. The inherent dynamics of this cellular immune response are not well understood for two major reasons: i. Type-I IFN activation underlies temporal and quantitative cell-to-cell heterogeneity vastly restricting the validity of cell population-level studies. ii. Experimental stimulation of cellular receptors that lead to IFN activation is either achieved via viral infection or via endocytic uptake of synthetic compounds. Both approaches result in heterogeneity in response timing and response magnitude within the treated/infected cell population. Using novel approaches comprising a dual reporter cell line (IRF-3-TagRFP, IFN-β-TurboGFP) and the electroporation of adherent cells together with extensive live-cell imaging, we identify dynamic properties of the central transcription factor IRF-3 at single-cell level upon stimulating the RIG-I receptor with synthetic dsRNA. IRF-3 shows surprising dynamical properties controlling initial and subsequent IFN-β responses in single cells. IRF-3 contributes to the randomisation of IFN-β expression rates between cells upon initial stimulation. Furthermore, our reporter cell line visualises concentration-dependent effects of IRF-3 activation and IFN-β expression and the impact of TNF-α and IFN-β pre-treatment. In summary, single-cell studies and defined intracellular receptor stimulation have unravelled response patterns and dynamic properties of the antiviral type-I IFN activation pathway that can now serve to characterise virus-activated responses in detail or design optimised vaccine adjuvants.

ID: 60: Novel intrinsic/innate pathways that sense the “stress” of virus infection

Directly sensing virus particles is critical as a first line of antiviral defense, because once allowed to replicate, most viruses will prevent induction of a response. While nucleic acid sensing is sufficient to recognize replicating virus, the genomic material within incoming virus particles is limited and often concealed by viral proteins. We previously found that cells sense membrane perturbation associated with enveloped virus fusion with cellular membranes, leading to induction of a modified, interferon-independent antiviral response. We recently discovered that membrane perturbation by diverse enveloped particles triggers Ca2+ signalling, an essential component of an antiviral response. In addition to Ca2+ signaling, sensing of incoming genomes requires either the cGAS-STING or RLR-MAVS signaling pathway. While viral nucleic acid alone is neither necessary nor sufficient to trigger an antiviral response, Ca2+ oscillations triggered by plasma membrane fusion is also insufficient. Thus, we hypothesize that Ca2+ plays a priming role for the recognition of genomic material within incoming particles, and that cells have evolved novel sensing pathways that recognize the “stress” of homeostasis dysregulation inherent to virus entry. Current studies are aimed at elucidating these novel pathways, particularly mechanisms of activating the key transcription factor IRF3 in the absence of nucleic acid sensing.