IFN Regulatory Factor Research Articles

Grouper IFIT1 inhibits iridovirus and nodavirus infection by positively regulating interferon response

Interferon-induced protein with tetratricopeptide repeats 1 (IFIT1), one of the interferon stimulated genes (ISGs), is strongly induced by type I interferon (IFN), double-stranded RNAs and virus infection. To investigate the actions of fish IFIT1 in response to virus infection, we cloned an IFIT1 homolog from orange spotted grouper (EcIFIT1) and clarified its function in this study. The full-length cDNA of EcIFIT1 is 1839 bp, which is composed of 436 amino acid (aa) residues, with 77.8% and 22.8% identity to IFIT1 homolog of yellow perch (Perca flavescens) and humans (homo sapiens), respectively. Sequence alignment analysis showed that EcIFIT1 contained three tetratricopeptide repeats (TPRs). Tissue distribution analysis indicated that EcIFIT1 was abundant in intestine, spleen, liver, and heart. Moreover, EcIFIT1 was significantly up-regulated by Singapore grouper iridovirus (SGIV) or red-spotted grouper nervous necrosis virus (RGNNV) infection, and polyinosinic-polycytidylic acid (poly I:C) or lipopolysaccharide (LPS) treatment in vitro. Under fluorescence microscopy, EcIFIT1 was found to localize throughout the cytoplasm in transfected cells. EcIFIT1 overexpression significantly suppressed the replication of SGIV and RGNNV, demonstrated by decreasing the cytopathic effect (CPE) severity, viral gene transcription and the virus titers. Further studies showed that the ectopic expression of EcIFIT1 increased the transcription level of IFN related molecules, including IFN regulatory factor (IRF) 3, IRF7, IFN stimulated gene (ISG) 15 and myxovirus resistance gene (MX) I. Meanwhile, the expression levels of pro-inflammation cytokines were differently regulated by the ectopic expression of EcIFIT1. In addition, flow cytometry analysis suggested that EcIFIT1 overexpression affected cell cycle progression by mediating S/G2 transition. Taken together, our results indicated that EcIFIT1 might exert antiviral function against fish virus by up-regulating interferon response or affecting cell cycle.

Human cytomegalovirus induces and exploits Roquin to counteract the IRF1-mediated antiviral state

RNA represents a pivotal component of host-pathogen interactions. Human cytomegalovirus (HCMV) infection causes extensive alteration in host RNA metabolism, but the functional relationship between the virus and cellular RNA processing remains largely unknown. Through loss-of-function screening, we show that HCMV requires multiple RNA-processing machineries for efficient viral lytic production. In particular, the cellular RNA-binding protein Roquin, whose expression is actively stimulated by HCMV, plays an essential role in inhibiting the innate immune response. Transcriptome profiling revealed Roquin-dependent global down-regulation of proinflammatory cytokines and antiviral genes in HCMV-infected cells. Furthermore, using cross-linking immunoprecipitation (CLIP)-sequencing (seq), we identified IFN regulatory factor 1 (IRF1), a master transcriptional activator of immune responses, as a Roquin target gene. Roquin reduces IRF1 expression by directly binding to its mRNA, thereby enabling suppression of a variety of antiviral genes. This study demonstrates how HCMV exploits host RNA-binding protein to prevent a cellular antiviral response and offers mechanistic insight into the potential development of CMV therapeutics.

Noncanonical Role of FBXO6 in Regulating Antiviral Immunity.

The evolutionarily conserved F-box family of proteins are well known for their role as the key component of SKP1-Cullin1-F-box (SCF) E3 ligase in controlling cell cycle, cell proliferation and cell death, carcinogenesis, and cancer metastasis. However, thus far, there is only limited investigation on their involvement in antiviral immunity. In contrast to the canonical function of FBXO6 associated with SCF E3 ligase complex, we report, in this study, that FBXO6 can also potently regulate the activation of IFN-I signaling during host response to viral infection by targeting the key transcription factor IFN-regulatory factor 3 (IRF3) for accelerated degradation independent of SCF in human embryonic kidney cells (HEK293T) and human lung cancer epithelial cells (A549). Structure and function delineation has further revealed that FBXO6 interacts with IAD domain of IRF3 through its FBA region to induce ubiquitination and degradation of IRF3 without the involvement of SCF. Thus, our studies have identified a general but, to our knowledge, previously unrecognized role and a novel noncanonical mechanism of FBXO6 in modulating IFN-I-mediated antiviral immune responses, which may protect the host from immunopathology of overreactive and harmful IFN-I production.

Characteristic genes in THP‑1 derived macrophages infected with Mycobacterium tuberculosis H37Rv strain identified by integrating bioinformatics methods.

Mycobacterium tuberculosis (M. tb) is a highly successful pathogen that has co-existed with humans for 1,000's of years. As the cornerstone of the immune system, macrophages are a key part of innate immunity. They ingest and degrade foreign substances including aging cells and microorganisms, coordinate the inflammatory process, and are the first line of defense against M. tb infection. Recent advances in cellular mycobacteriology have indicated that M. tb uses an remarkably complex strategy to disrupt macrophage function, in order to counteract the antimicrobial mechanisms of the innate and adaptive immune responses, thereby achieving immune escape. With the popularity of microarray technology, a variety of public platforms have provided a variety of gene expression data associated with physiological and disease conditions. Meta-analysis can systematically and quantitatively analyze multiple independent data concerning the same disease, greatly improving the statistical significance and credibility of the gene expression data analysis performed. In the present study, 6 microarray expression datasets of human acute monocytic leukemia THP-1 cell line infected by M. tb H37Rv strain were collected from the GEO database. A total of 4 high-quality datasets were identified using meta-analysis methods in R language, and 306 differentially expressed genes with statistical significance were obtained. Then, a protein-protein interaction (PPI) network of these differentially expressed genes was constructed on the Search Tool for the Retrieval of Interacting Genes/Proteins Database online tool and visualized by Cytoscape v. 3.6.1 software. Using CentiScape and MCODE plugin in the Cytoscape software to mine the functional modules associated with M. tb infection process, 32 characteristic genes were identified. Gene ontology and Kyoto Encyclopedia of Genes and Genomes analysis was performed on the 32 characteristic genes, and it was demonstrated that these genes were primarily associated with the type I interferon (IFN) pathway. In the established model of THP-1-derived macrophages infected by M. tb, the actual differential expression levels of IFN-stimulated gene 15 (ISG15), 2′-5-oligoadenylate synthetase like (OASL), IFN regulatory factor 7 (IRF7) and DExD/H-box helicase 58 (DDX58), the first 4 genes of the 32 characteristic genes, were verified by reverse transcription quantitative polymerase chain reaction. The results were consistent with the results of microarray analysis. The association between ISG15, OASL and IRF7 and TB infection was also verified. Although a number of studies have identified that the type I IFN pathway may assist M. tb to achieve immune escape, the present study used a meta-analysis of microarray data and PPI network analysis to examine some of the novel genes identified in the IFN pathway. The results furthered the understanding of the molecular mechanisms of the TB immune response and provided a novel perspective for future therapeutic goals.

Immunobiotic Strains Modulate Toll-Like Receptor 3 Agonist Induced Innate Antiviral Immune Response in Human Intestinal Epithelial Cells by Modulating IFN Regulatory Factor 3 and NF-κB Signaling.

Many studies have demonstrated that immunobiotics with immunoregulatory functions improve the outcomes of several bacterial and viral infections by modulating the mucosal immune system. However, the precise mechanisms underlying the immunoregulatory and antiviral activities of immunobiotics have not yet been elucidated in detail. The present study was conducted to determine whether selected lactic acid bacteria (LAB) modulate toll-like receptor 3 (TLR3) agonist polyinosinic:polycytidylic acid (PolyI:C) induced viral response in human intestinal epithelial cells (IECs). PolyI:C increased the expression of interferon-β (IFN-β), interleukin-6 (IL-6), interleukin-8 (IL-8), monocyte chemoattractant protein (MCP-1), and interleukin-1β (IL-1β) in HCT116 cells, and these up-regulations were significantly altered when cells were pre-stimulated with LAB isolated from Korean fermented foods. LAB strains were capable to up-regulate IFN-β but down-regulated IL-6, IL-8, MCP-1, and IL-1β mRNA levels as compared with PolyI: C. HCT-116 cell treatment with LABs beneficially modulated the mRNA levels of C-X-C motif chemokine 10 (CXCL-10), 2-5A oligoadenylate synthetase 1 (OSA1), myxovirus resistance protein (MxA), TLR3, and retinoic acid inducible gene-I (RIG-I), and TLR negative regulators. In addition, LABs increased IFN-β, IFN-α, and interleukin-10 (IL-10) and decreased tumor necrosis factor-α (TNF-α) and IL-1β protein/mRNA levels in THP-1 cells. LABs also protected the cells by maintaining tight-junction proteins (zonula occludens-1 and occludin). The beneficial effects of these LABs were mediated via modulation of the interferon regulatory factor 3 (IRF3) and nuclear factor-kappa B (NF-κB) pathways. Overall, the results of this study indicate that immunobiotics have potent antiviral and anti-inflammatory activities that may use as antiviral substitutes for human and animal applications.

Microbiota Metabolite Short-Chain Fatty Acids Facilitate Mucosal Adjuvant Activity of Cholera Toxin through GPR43.

The gut microbiota has been shown critical for mucosal adjuvant activity of cholera toxin (CT), a potent mucosal adjuvant. However, the mechanisms involved remain largely unknown. In this study, we report that depletion of gut bacteria significantly decreased mucosal and systemic Ab responses in mice orally immunized with OVA and CT. Feeding mice short-chain fatty acids (SCFAs) promoted Ab responses elicited by CT, and, more importantly, rescued Ab responses in antibiotic-treated mice. In addition, mice deficient in GPR43, a receptor for SCFAs, showed impaired adjuvant activity of CT. Administering CT did not promote SCFA production in the intestines; thus, SCFAs facilitated but did not directly mediate the adjuvant activity of CT. SCFAs promoted B cell Ab production by promoting dendritic cell production of BAFF and ALDH1a2, which induced B cell expression of IFN regulatory factor 4, Blimp1, and XBP1, the plasma B cell differentiation-related genes. Furthermore, when infected with Citrobacter rodentium, GPR43-/- mice exhibited decreased Ab responses and were more susceptible to infection, whereas the administration of SCFAs promoted intestinal Ab responses in wild-type mice. Our study thereby demonstrated a critical role of gut microbiota and their metabolite SCFAs in promoting mucosal adjuvant activity of CT through GPR43.

CRISPR/Cas9-based Knockout Strategy Elucidates Components Essential for Type 1 Interferon Signaling in Human HeLa Cells

Type I interferons (IFNs) have a central role in innate and adaptive immunities, proliferation, and cancer surveillance. How IFN binding to its specific receptor, the IFN α and β receptor (IFNAR), can drive such variety of processes is an open question. Here, to systematically and thoroughly investigate the molecular mechanism of IFN signaling, we used a CRISPR/Cas9-based approach in a human cell line (HeLa) to generate knockouts (KOs) of the genes participating in the type 1 IFN signaling cascade. We show that both IFNAR chains (IFNAR1 and IFNAR2) are absolutely required for any IFN-induced signaling. Deletion of either signal transducer and activator of transcription 1 (STAT1) or STAT2 had only a partial effect on IFN-induced antiviral activity or gene induction. However, the deletion of both genes completely abrogated any IFN-induced activity. So did a double STAT2–IFN regulatory factor 1 (IRF1) KO and, to a large extent, a STAT1 KO together with IRF9 knockdown. KO of any of the STATs had no effect on the phosphorylation of other STATs, indicating that they bound IFNAR independently. STAT3 and STAT6 phosphorylations were fully induced by type 1 IFN in the STAT1–STAT2 KO, but did not promote gene induction. Moreover, STAT3 KO did not affect type 1 IFN-induced gene or protein expression. Type 1 IFN also did not activate p38, AKT, or ERK kinase. We conclude that type 1 IFN-induced activities in HeLa cells are mediated by STAT1/STAT2/IRF9, STAT1/STAT1, or STAT2/IRF9 complexes and do not require alternative pathways.

Zika virus circumvents host innate immunity by targeting the adaptor proteins MAVS and MITA

Recently, Zika virus (ZIKV) has generated extraordinary concern because of its severe neurotoxicity. Disturbingly, there is no vaccine or specific drug to prevent or treat the diseases caused by ZIKV infection. Thus, it is extremely urgent to characterize the pathogenesis of ZIKV. It has been documented that ZIKV can evade antiviral responses of host cells. Here, we demonstrate that ZIKV strain SZ-WIV01 down-regulates the production of type I IFN and IFN-stimulated genes along with the expression of mitochondrial antiviral signaling protein (MAVS) and mediator of IFN regulatory factor 3 activation (MITA). In the mechanism, ZIKV nonstructural (NS) 3 and NS2B3 negatively regulate IFN-related retinoic acid-inducible gene I-like receptor signaling pathway by targeting MAVS and MITA, respectively. Overexpression of ZIKV NS3 and NS2B3 dramatically inhibits expression of IFN-β. ZIKV NS3 interacts with MAVS, and NS2B3 interacts with MITA, which catalyzes K48-linked polyubiquitination of MAVS and MITA for degradation. Further investigations suggest that ZIKV NS2B3 impairs polyinosinic:polycytidylic acid-triggered K63-linked polyubiquitination of MITA, thereby subverting the activation of downstream sensors. Our study reveals an undiscovered mechanism for ZIKV to escape the innate immune response, providing new insights into clinical study of vaccines or effective drugs.-Li, W., Li, N., Dai, S., Hou, G., Guo, K., Chen, X., Yi, C., Liu, W., Deng, F., Wu, Y., Cao, X. Zika virus circumvents host innate immunity by targeting the adaptor proteins MAVS and MITA.

Janus kinase 1/2 inhibition for the treatment of autoinflammation associated with heterozygous TNFAIP3 mutation

Janus kinase 1/2 inhibition for the treatment of autoinflammation associated with heterozygous TNFAIP3 mutation

A New IRF-1-Driven Apoptotic Pathway Triggered by IL-4/IL-13 Kills Neonatal Th1 Cells and Weakens Protection against Viral Infection.

Early life immune responses are deficient in Th1 lymphocytes that compromise neonatal vaccination. We found that IL-4 and IL-13 engage a developmentally expressed IL-4Rα/IL-13Rα1 heteroreceptor to endow IFN regulatory factor 1 (IRF-1) with apoptotic functions, which redirect murine neonatal Th1 reactivation to cell death. IL-4/IL-13-induced STAT6 phosphorylation serves to enhance IRF-1 transcription and promotes its egress from the nucleus. In the cytoplasm, IRF-1 can no longer serve as an anti-viral transcription factor but, instead, colocalizes with Bim and instigates the mitochondrial, or intrinsic, death pathway. The new pivotal function of IRF-1 in the death of neonatal Th1 cells stems from the ability of its gene to bind STAT6 for enhanced transcription and the proficiency of its protein to precipitate Bim-driven apoptosis. This cytokine-induced, IRF-1-mediated developmental death network weakens neonatal Th1 responses during early life vaccination and increases susceptibility to viral infection.

Patients with tumour necrosis factor (TNF) receptor-associated periodic syndrome (TRAPS) are hypersensitive to Toll-like receptor 9 stimulation.

Tumour necrosis factor receptor-associated periodic syndrome (TRAPS) is a hereditary autoinflammatory disorder characterized by recurrent episodes of fever and inflammation. It is associated with autosomal dominant mutations in TNFRSF1A, which encodes tumour necrosis factor receptor 1 (TNF-R1). Our aim was to understand the influence of TRAPS mutations on the response to stimulation of the pattern recognition Toll-like receptor (TLR)-9. Peripheral blood mononuclear cells (PBMCs) and serum were isolated from TRAPS patients and healthy controls: serum levels of 15 proinflammatory cytokines were measured to assess the initial inflammatory status. Interleukin (IL)-1β, IL-6, IL-8, IL-17, IL-22, tumour necrosis factor (TNF)-α, vascular endothelial growth factor (VEGF), interferon (IFN)-γ, monocyte chemoattractant protein 1 (MCP-1) and transforming growth factor (TGF)-β were significantly elevated in TRAPS patients' sera, consistent with constitutive inflammation. Stimulation of PBMCs with TLR-9 ligand (ODN2006) triggered significantly greater up-regulation of proinflammatory signalling intermediates [TNF receptor-associated factor (TRAF 3), IL-1 receptor-associated kinase-like 2 (IRAK2), Toll interacting protein (TOLLIP), TRAF6, phosphorylated transforming growth factor-β-activated kinase 1 (pTAK), transforming growth factor-β-activated kinase-binding protein 2 (TAB2), phosphorylated TAK 2 (pTAB2), IFN-regulatory factor 7 (IRF7), receptor interacting protein (RIP), nuclear factor kappa B (NF-κB) p65, phosphorylated NF-κB p65 (pNF-κB p65) and mitogen-activated protein kinase kinase (MEK1/2)] in TRAPS patients' PBMCs. This up-regulation of proinflammatory signalling intermediates and raised serum cytokines occurred despite concurrent anakinra treatment and no overt clinical symptoms at time of sampling. These novel findings further demonstrate the wide-ranging nature of the dysregulation of innate immune responses underlying the pathology of TRAPS and highlights the need for novel pathway-specific therapeutic treatments for this disease.

Human β-defensin 2 is involved in CCR2-mediated Nod2 signal transduction, leading to activation of the innate immune response in macrophages

Beta-defensins contribute to host innate defense against various pathogens, including viruses, although the details of their roles in innate immune cells are unclear. We previously reported that human β-defensin 2 (HBD 2) activates primary innate immunity against viral infection and suggested that it plays a role in the induction of the adaptive immune response. We analyzed the mechanisms by which HBD 2 primes innate antiviral immunity and polarized activation of macrophage-like THP-1 cells using the receptor-binding domain (RBD) of Middle East respiratory syndrome coronavirus (MERS-CoV) spike protein (S RBD) as a model antigen. The expression of nucleotide-binding oligomerization domain containing 2 (Nod2), type I interferons, (IFNs), and proinflammatory mediators was enhanced in S RBD-HBD 2-treated THP-1 cells. S RBD-HBD 2 treatment also enhanced phosphorylation and activation of receptor-interacting serine/threonine-protein kinase 2 and IFN regulatory factor 3 compared to S RBD alone. Finally, HBD 2-conjugated S RBD interacted with C-C chemokine receptor 2 (CCR2), and Nod2 was involved in HBD 2-mediated CCR2 signaling, which was associated with the activation and M1 polarization of THP-1 cells. Therefore, HBD 2 promotes CCR2-mediated Nod2 signaling, which induces production of type I IFNs and an inflammatory response, and enhances primary innate immunity leading to an effective adaptive immune response to HBD 2-conjugated antigen.

GM-CSF- and IRF4-Dependent Signaling Can Regulate Myeloid Cell Numbers and the Macrophage Phenotype during Inflammation.

Studies have demonstrated the importance of a GM-CSF→IFN regulatory factor 4 (IRF4)→CCL17 pathway, first identified in monocytes/macrophages, for arthritic pain and disease development. In this study, we further investigated the involvement of this new pathway in shaping the inflammatory response using the zymosan-induced peritonitis (ZIP) model. ZIP (8 mg of zymosan, i.p., day 0) was induced in C57BL/6 wild-type (WT), GM-CSF-/- , Irf4-/- , and Ccl17E/E mice. In comparison with WT mice, GM-CSF-/- and Irf4-/- mice had a reduced ZIP response, as judged by a reduced number of neutrophils and macrophages in the peritoneal cavity. Moreover, the phenotype of the ZIP macrophages was altered by a lack of GM-CSF or IRF4 (increased IL-10 secretion and Arg1 mRNA expression), with IRF4 levels being lower in GM-CSF-/- ZIP macrophages than in the WT cells. In addition, GM-CSF ̶IRF4 signaling upregulated MHC class II expression in ZIP macrophages and bone marrow-derived macrophages. Although Ccl17 mRNA expression was reduced in ZIP macrophages in the absence of either GM-CSF or IRF4, thus supporting the presence of the new pathway in inflammatory macrophages, CCL17 did not modulate the inflammatory response, both in terms of number of myeloid cells or the macrophage phenotype. Thus, during an inflammatory response, both macrophage numbers and their phenotype can depend on GM-CSF- and IRF4-dependent signaling independently of CCL17.

Brucella abortus Cyclic Dinucleotides Trigger STING-Dependent Unfolded Protein Response That Favors Bacterial Replication.

Brucella abortus is a facultative intracellular bacterium that causes brucellosis, a prevalent zoonosis that leads to abortion and infertility in cattle, and undulant fever, debilitating arthritis, endocarditis, and meningitis in humans. Signaling pathways triggered by B. abortus involves stimulator of IFN genes (STING), which leads to production of type I IFNs. In this study, we evaluated the pathway linking the unfolded protein response (UPR) and the endoplasmic reticulum-resident transmembrane molecule STING, during B. abortus infection. We demonstrated that B. abortus infection induces the expression of the UPR target gene BiP and XBP1 in murine macrophages through a STING-dependent pathway. Additionally, we also observed that STING activation was dependent on the bacterial second messenger cyclic dimeric GMP. Furthermore, the Brucella-induced UPR is crucial for induction of multiple molecules linked to type I IFN signaling pathway, such as IFN-β, IFN regulatory factor 1, and guanylate-binding proteins. Furthermore, IFN-β is also important for the UPR induction during B. abortus infection. Indeed, IFN-β shows a synergistic effect in inducing the IRE1 axis of the UPR. In addition, priming cells with IFN-β favors B. abortus survival in macrophages. Moreover, Brucella-induced UPR facilitates bacterial replication in vitro and in vivo. Finally, these results suggest that B. abortus-induced UPR is triggered by bacterial cyclic dimeric GMP, in a STING-dependent manner, and that this response supports bacterial replication. In summary, association of STING and IFN-β signaling pathways with Brucella-induced UPR unravels a novel link between innate immunity and endoplasmic reticulum stress that is crucial for bacterial infection outcome.

IRF2BP2: A new player in the regulation of cell homeostasis.

The IRF2BP2 (IFN regulatory factor 2 binding protein 2) protein was identified as a nuclear protein that interacts with IFN regulatory factor 2 (IRF-2) and is an IRF-2-dependent transcriptional repressor. IRF2BP2 belongs to the IRF2BP family, which includes IRF2BP1, IRF2BP2, and IRF2BPL (EAP1). Recently, IRF2BP2 has emerged as an important new transcriptional cofactor in different biological systems, acting as a positive and negative regulator of gene expression. IRF2BP2 plays a role in different cellular functions, including apoptosis, survival, and cell differentiation. Additionally, IRF2BP2 may be involved in cancer development. Finally, it has been recently reported that IRF2BP2 may play a role in macrophage regulation and lymphocyte activation, highlighting its function in innate and adaptive immune responses. However, it has become increasingly clear that IRF2BP2 and its isoforms can have specific functions. In this review, we address the possible reasons for these distinct roles of IRF2BP2 and the partner proteins that interact with it. We also discuss the genes regulated by IRF2BP2 during the immune response and in other biological systems.

Nonreceptor Tyrosine Kinase c-Abl- and Arg-Mediated IRF3 Phosphorylation Regulates Innate Immune Responses by Promoting Type I IFN Production.

The nonreceptor tyrosine kinase c-Abl plays important roles in T cell development and immune responses; however, the mechanism is poorly understood. IFN regulatory factor 3 (IRF3) is a key transcriptional regulator of type I IFN-dependent immune responses against DNA and RNA viruses. The data in this study show that IRF3 is physically associated with c-Abl in vivo and directly binds to c-Abl in vitro. IRF3 is phosphorylated by c-Abl and c-Abl-related kinase, Arg, mainly at Y292. The inhibitor AMN107 inhibits IFN-β production induced by poly(dA:dT), poly(I:C), and Sendai virus in THP-1 and mouse bone marrow-derived macrophage cells. IRF3-induced transcription of IFN-β is significantly reduced by the mutation of Y292 to F. Moreover, AMN107 suppresses gene expression of absent in melanoma 2 (AIM2) and subsequently reduces inflammasome activation induced by cytosolic bacteria, dsDNA, and DNA viruses. Consistent with this finding, Francisella tularensis subsp. holarctica live vaccine strain (Ft LVS), which is known as an activator of AIM2 inflammasome, induces death in significantly more C57BL/6 mice treated with the Abl inhibitor AMN107 or c-Abl/Arg small interfering RNA than in untreated mice. This study provides new insight into the function of c-Abl and Arg in regulating immune responses and AIM2 inflammasome activation, especially against Ft LVS infection.

Structural mechanism of vaccinia virus protein C6 mediated inactivation of transcription factors IRF3 and IRF7 via inhibition of TBK1 mediated scaffold protein‐protein interactions

Interferons (IFNs) are signaling proteins with potent antiviral and immune stimulatory effects. IFN signaling is initiated after host cell pattern recognition receptors (PRRs) recognize pathogen‐associated molecular patterns (PAMPs) present in many viral proteins. This event in turn generates a complex signaling cascade involving numerous proteins, activation of various transcription factors (TF), such as IFN‐regulatory factors (IRF) 3 and 7. These TFs subsequently translocate to the nucleus to elevate the transcription of type I IFNs, cytokines, and chemokines. Viruses such as Vaccinia virus (VACV) have evolved many strategies to interfere with the IFNs or their mechanism of action. Specifically, VACV encodes a small 16kDa protein, C6, that inhibits the activation of IRF3/7 via binding with an upstream kinase, TBK1, and its adaptor proteins TANK, NAP1, and SINTBAD in order to interfere with the production of IFNβ. Binding with these adaptor proteins, C6 prevents TBK1/IKKɛ‐dependent activation of IRF3 and IRF7 allowing VACV to evade immune recognition. To elucidate the structural mechanism of how C6 mediated inhibition of these adaptor protein/TBK1 interactions can block the activation of IRF3 and IRF7, we have chosen C6 and TANK as canonical binding partners. We have identified the binding regions between C6 and TANK by utilizing co‐expression/co‐purification studies of C6 and TANK proteins, and co‐crystallization trials are ongoing. The immune evasive strategy exploited by VACV can be used to develop novel therapies, such as C6 mimetics to provide TBK1 inhibition. The development of therapeutics depends immensely on a detailed structural analysis of the interactions between C6 and adaptor proteins. Our structural studies of C6 and TBK1 adaptor proteins will provide useful insights in developing superior therapies against malignancies, infectious diseases, and autoimmune disorders.This abstract is from the Experimental Biology 2019 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.

15-Epi-LXA4 and MaR1 counter inflammation in stromal cells from patients with Achilles tendinopathy and rupture.

Resolution of inflammation is poorly understood in Achilles tendon disorders. Herein, we investigated the bioactive lipid mediator profiles of tendon-derived stromal cells isolated from patients with Achilles tendinopathy (AT) or Achilles rupture (AR) under baseline and IL-1β–stimulated conditions. We also determined whether incubating these cells with 2 of the mediators produced by tendon-derived stromal cells, 15-epi-Lipoxin A4 (15-epi-LXA4) or maresin (MaR)-1, moderated their proinflammatory phenotype. Under baseline conditions, AT cells showed concurrent increased levels of proinflammatory eicosanoids and proresolving mediators compared with AR cells. IL-1β treatment induced profound prostaglandin E2 release in AR compared with AT cells. Incubation of IL-1β treated AT and AR tendon-derived stromal cells in 15-epi-LXA4 or MaR1 reduced proinflammatory eicosanoids and potentiated the release of proresolving mediators. These mediators also induced specialized proresolving mediator (SPM) biosynthetic enzymes arachidonate lipoxygenase (ALOX) 12 and ALOX15 and up-regulated the proresolving receptor ALX compared with vehicle-treated cells. Incubation in 15-epi-LXA4 or MaR1 also moderated the proinflammatory phenotype of AT and AR cells, regulating podoplanin, CD90, signal transducer and activator of transcription (STAT)-1, IL-6, IFN regulatory factor (IRF) 5, and TLR4 and suppressed c-Jun N-terminal kinase 1/2/3, Lyn, STAT-3, and STAT-6 phosphokinase signaling. In summary, we identify proresolving mediators that are active in AT and AR and propose SPMs, including 15-epi-LXA4 or MaR1, as a potential strategy to counterregulate inflammatory processes in these cells.—Dakin, S. G., Colas, R. A., Newton, J., Gwilym, S., Jones, N., Reid, H. A. B., Wood, S., Appleton, L., Wheway, K., Watkins, B., Dalli, J., Carr, A. J. 15-Epi-LXA4 and MaR1 counter inflammation in stromal cells from patients with Achilles tendinopathy and rupture.

Rhesus Macaque Rhadinovirus Encodes a Viral Interferon Regulatory Factor To Disrupt Promyelocytic Leukemia Nuclear Bodies and Antagonize Type I Interferon Signaling.

Interferon (IFN) production and the subsequent induction of IFN-stimulated genes (ISGs) are highly effective innate strategies utilized by cells to protect against invading pathogens, including viruses. Critical components involved in this innate process are promyelocytic leukemia nuclear bodies (PML-NBs), which are subnuclear structures required for the development of a robust IFN response. As such, PML-NBs serve as an important hurdle for viruses to overcome to successfully establish an infection. Both Kaposi's sarcoma-associated herpesvirus (KSHV) and the closely related rhesus macaque rhadinovirus (RRV) are unique for encoding viral homologs of IFN regulatory factors (termed vIRFs) that can manipulate the host immune response by multiple mechanisms. All four KSHV vIRFs inhibit the induction of IFN, while vIRF1 and vIRF2 can inhibit ISG induction downstream of the IFN receptor. Less is known about the RRV vIRFs. RRV vIRF R6 can inhibit the induction of IFN by IRF3; however, it is not known whether any RRV vIRFs inhibit ISG induction following IFN receptor signaling. In our present study, we demonstrate that the RRV vIRF R12 aids viral replication in the presence of the type I IFN response. This is achieved in part through the disruption of PML-NBs and the inhibition of robust ISG transcription.IMPORTANCE KSHV and RRV encode a unique set of homologs of cellular IFN regulatory factors, termed vIRFs, which are hypothesized to help these viruses evade the innate immune response and establish infections in their respective hosts. Our work elucidates the role of one RRV vIRF, R12, and demonstrates that RRV can dampen the type I IFN response downstream of IFN signaling, which would be important for establishing a successful infection in vivo.

Myeloid TBK1 Signaling Contributes to the Immune Response to Influenza.

Macrophages provide key elements of the host response to influenza A virus (IAV) infection, including expression of type I IFN and inflammatory cytokines and chemokines. TBK1 (TNF receptor-associated factor family member-associated NF-κB activator-binding kinase 1) contributes to IFN expression and antiviral responses in some cell types, but its role in the innate response to IAV in vivo is unknown. We hypothesized that macrophage TBK1 contributes to both IFN and non-IFN components of host defense and IAV pathology. We generated myeloid-conditional TBK1 knockout mice and assessed the in vitro and in vivo consequences of IAV infection. Myeloid-specific loss of TBK1 in vivo resulted in less severe host response to IAV, as assessed by decreased mortality, weight loss, and hypoxia and less inflammatory changes in BAL fluid relative to wild-type mice despite no differences in viral load. Mice lacking myeloid TBK1 showed less recruitment of CD64+SiglecF-Ly6Chi inflammatory macrophages, less expression of inflammatory cytokines in the BAL fluid, and less expression of both IFN regulatory factor and NF-κB target genes in the lung. Analysis of sorted alveolar macrophages, inflammatory macrophages, and lung interstitial macrophages revealed that each subpopulation requires TBK1 for distinct components of the response to IAV infection. Our findings define roles for myeloid TBK1 in IAV-induced lung inflammation apart from IFN type I expression and point to myeloid TBK1 as a central and cell type-specific regulator of virus-induced lung damage.