Interferon-stimulated Response Element Research Articles

The response of hepatitis B virus genotype to interferon is associated with a mutation in the interferon-stimulated response element.

Genetic variation and genotype of Hepatitis B virus (HBV) are related to the efficiency of interferon alpha (IFN-α)-based antiviral therapy. However, the correlation of variation in interferon-stimulated response element (ISRE) and HBV genotype response to IFN-α therapy remains elusive.Differences of ISRE between genotype B and C HBV were explored using the HBV sequences retrieved from GenBank, and further investigated by ISRE region cloning and sequencing from 60 clinical samples post-IFN-α therapy. Additionally, ISRE mutants were constructed and their relation to responsiveness of IFN-α was evaluated by real-time PCR and Southern blot analysis.ISRE pattern between genotype B and C were found based on both clinical sample sequencing and full-length sequence alignment. The primary difference is the fourth base within the ISRE region, with T and C for genotype B and C, respectively. HBV with genotype C-type ISRE had a higher replicative capability as compared to HBV with genotype B-type ISRE after IFN-α treatment in huh7 cells.Conclusion:Preference of ISRE between genotype B and C HBV are distinct. Single nucleotide difference (C to T) within the HBV ISRE region may link to the efficacy of IFN-α therapy to genotype B and C HBV. Therefore, this study provides a clue for the determination of IFN-α therapy response to HBV treatment.

Genome-Wide Classification of Type I, Type II and Type III Interferon-Stimulated Genes in Chicken Fibroblasts.

Interferons (IFNs) play central roles in establishing innate immunity and mediating adaptive immunity against multiple pathogens. Three known types of IFNs identify their cognate receptors, initiate cascades of signalling events and eventually result in the induction of a myriad of IFN-stimulated genes (ISGs). These ISGs perform a multitude of functions and cumulatively corroborate a bespoke antiviral state to safeguard hosts against invading viruses. Owing to the unique nature of a chicken’s immune system and the lack of foundational profiling information on the nature and dynamic expression of IFN-specific ISGs at the genome scale, we performed a systematic and extensive analysis of type I, II and III IFN-induced genes in chicken. Employing pan-IFN responsive chicken fibroblasts coupled with transcriptomics, we observed an over-representation of up-regulated ISGs compared to down-regulated ISGs by all types of IFNs. Intriguingly, prediction of IFN-stimulated response element (ISRE) and gamma-IFN activation sequence (GAS) revealed a substantial number of GAS motifs in selective and significantly induced ISGs in chicken. Extensive comparative, genome-wide and differential expression analysis of ISGs under equivalent signalling input catalogue a set of genes that were either IFN-specific or independent of types of IFNs used to prime fibroblasts. These comprehensive datasets, first of their kinds in chicken, will establish foundations to elucidate the mechanisms of actions and breadth of antiviral action of ISGs, which may propose alternative avenues for targeted antiviral therapy against viruses of poultry of public health importance.

Genetic Polymorphisms within Interferon-λ Region and Interferon-λ3 in the Human Pathophysiology: Their Contribution to Outcome, Treatment, and Prevention of Infections with Hepatotropic Viruses.

Genetic polymorphisms within the interferon λ (IFN-λ) chromosomal region, mainly rs12979860 of IFN-λ4 gene (IFNL4), are known as associated with spontaneous hepatitis C virus (HCV) resolution and sustained viral response to therapy with pegylated interferon- α and ribavirin. Strong linkage disequilibrium of IFNL4 rs12979860 with IFNL4 rs368234815, which is casually associated with HCV spontaneous and therapeutical eradication, at least partially explains favorable HCV outcomes attributed to major homozygosity in rs12979860. Effects of IFN-based antiviral treatment are associated with pretreatment expression of the IFN-λ1 receptor, expression of hepatic IFN-stimulated genes, production of IFN- λ4, and preactivation of the JAK-STAT signaling. Nowadays direct-acting antivirals (DAAs) became a potent tool in the treatment of hepatitis C, but IFN-λs are still under investigation as potential antivirals and might be an option in HCV infection (DAA resistance, recurrent viremia, adverse effects). Patients with altered immunocompetence are especially prone to infections. In uremic subjects, polymorphisms within the IFN-λ chromosomal region associate with spontaneous HCV clearance, similarly like in the non-uremic population. Circulating IFN-λ3 shows a positive correlation with plasma titers of antibodies to surface antigen of hepatitis B virus (anti-HBs), which are crucial for protection against hepatitis B virus. More efficient anti-HBs production in the presence of higher IFN-λ3 levels might occur due to IFN-λ3-induced regulation of indoleamine 2,3-dioxygenase (IDO) expression. IFN-stimulated response element is a part of IDO gene promoter. It is worth further investigation whether IDO gene, circulating IDO, genetic polymorphisms within the IFN-λ region, and circulating IFN-λ3 act in concordance in immunological response to hepatotropic viruses.

Nuclear Sensor Interferon-Inducible Protein 16 Inhibits the Function of Hepatitis B Virus Covalently Closed Circular DNA by Integrating Innate Immune Activation and Epigenetic Suppression.

Nuclear-located covalently closed circular DNA (cccDNA) of hepatitis B virus (HBV) is a determining factor for HBV persistence and the key obstacle for a cure of chronic hepatitis B. However, it remains unclear whether and how the host immune system senses HBV cccDNA and its biological consequences. Here, we demonstrated that interferon-inducible protein 16 (IFI16) could serve as a unique innate sensor to recognize and bind to HBV cccDNA in hepatic nuclei, leading to the inhibition of cccDNA transcription and HBV replication. Mechanistically, our data showed that IFI16 promoted the epigenetic suppression of HBV cccDNA by targeting an interferon-stimulated response element (ISRE) present in cccDNA. It is of interest that this ISRE was also revealed to play an important role in IFI16-activated type I interferon responses. Furthermore, our data revealed that HBV could down-regulate the expression level of IFI16 in hepatocytes, and there was a negative correlation between IFI16 and HBV transcripts in liver biopsies, suggesting the possible role of IFI16 in suppressing cccDNA function under physiological conditions. The nuclear sensor IFI16 suppresses cccDNA function by integrating innate immune activation and epigenetic regulation by targeting the ISRE of cccDNA, and IFI16 may present as a therapeutic target against HBV infection.

Structure-Activity Relationship Studies To Identify Affinity Probes in Bis-aryl Sulfonamides That Prolong Immune Stimuli.

Agents that safely induce, enhance, or sustain multiple innate immune signaling pathways could be developed as potent vaccine adjuvants or coadjuvants. Using high-throughput screens with cell-based nuclear factor κB (NF-κB) and interferon stimulating response element (ISRE) reporter assays, we identified a bis-aryl sulfonamide bearing compound 1 that demonstrated sustained NF-κB and ISRE activation after a primary stimulus with lipopolysaccharide or interferon-α, respectively. Here, we present systematic structure-activity relationship (SAR) studies on the two phenyl rings and amide nitrogen of the sulfonamide group of compound 1 focused toward identification of affinity probes. The murine vaccination studies showed that compounds 1 and 33 when used as coadjuvants with monophosphoryl lipid A (MPLA) showed significant enhancement in antigen ovalbumin-specific immunoglobulin responses compared to MPLA alone. SAR studies pointed to the sites on the scaffold that can tolerate the introduction of aryl azide, biotin, and fluorescent rhodamine substituents to obtain several affinity and photoaffinity probes which will be utilized in concert for future target identification and mechanism of action studies.

Polymorphisms of the Chicken Mx Gene Promoter and Association with Chicken Embryos' Susceptibility to Virulent Newcastle Disease Virus Challenge.

Newcastle disease is a devastating viral disease of chicken in low- and middle-income countries where the backyard production system is predominant. Marker-assisted selection of chickens that are resistant to Newcastle disease virus (NDV) is the promising strategy that needs to be explored. The aim of the present study was to investigate polymorphisms of the promoter region of the chicken Mx gene and association with Kuroiler, Sasso, and local Tanzanian chicken embryos' survival variability to virulent NDV infection. Chicken embryos were initially challenged with a minimum lethal dose of virulent NDV suspension and then were followed over time to gather information on their survival variability. Using the survival data, high and less susceptible cohorts were established, and a total of 88 DNA samples from high and less susceptible groups were genotypes by sequencing. Five single-nucleotide polymorphisms (SNPs), which were previously reported, were detected. Interestingly, for the first time, the findings demonstrated the association of the promoter region of chicken myxovirus-resistance (Mx) gene polymorphisms with chicken embryos' susceptibility to the virulent NDV challenge. At the genotypic level, the SNP4 G > A mutation that was located within the IFN-stimulating response element was associated (LR: 6.97, P=0.03) with chicken embryos' susceptibility to the virulent NDV challenge. An allele G frequency was higher in the less susceptible cohort, whereas an allele A frequency was higher in the high susceptible cohort. At the haplotype level, the haplotype group ACGC was associated (OR: 9.8, 95% CI: 1.06–79.43, P=0.042) with the same trait and had a resistant effect. In conclusion, the results have demonstrated the association of chicken Mx gene promoter polymorphisms and chicken embryos' survival variability to the virulent NDV challenge, and the information is useful for breeding programs designed to develop chicken genotypes that are resistant to Newcastle disease virus.

Molecular cloning, expression and functional analysis of Atg16L1 from orange-spotted grouper (Epinephelus coioides)

Autophagy related gene 16 (Atg16), which encodes a core protein for autophagosome formation, participates in autophagy activity, the ubiquitin proteasome system and inflammatory response in mammals. In this study, we cloned and characterized an Atg16 homolog from orange-spotted grouper (Epinephelus coioides) (EcAtg16L1). EcAtg16L1 encodes a 656-amino acid polypeptide, which shares 94.22% and 72.65% homology with large yellow croakers (Larimichthys crocea) and humans (Homo sapiens), respectively. EcAtg16L1 contains a conserved Atg16 domain and a WD-repeat-containing domain. Subcellular localization showed that EcAtg16L1 was distributed in the cytoplasm of grouper cells with a dot-like pattern. EcAtg16L1 overexpression promoted Singapore grouper iridovirus (SGIV) and red-spotted grouper nervous necrosis virus (RGNNV) replication, as evidenced by the increase in viral gene transcription and viral coat protein. Furthermore, EcAtg16L1 overexpression negatively regulated interferon (IFN)-related molecules and proinflammatory cytokines, and decreased IFN, IFN-stimulated response element, and nuclear factor κB promoter activities. Taken together, aside from its function in autophagosome formation, EcAtg16L1 also plays role in promoting SGIV and RGNNV replication and the pro-viral effect might involve its down regulation to interferon and inflammatory responses.

Grouper Atg12 negatively regulates the antiviral immune response against Singapore grouper iridovirus (SGIV) infection

Autophagy is an evolutionarily conserved, multi-step lysosomal degradation process used to maintain cell survival and homeostasis. A series of autophagy-related genes (Atgs) are involved in the autophagic pathway. In mammals, a growing number of studies have attributed functions to some Atgs that are distinct from their classical role in autophagosome biogenesis, such as resistance to pathogens. However, little is known about the functions of fish Atgs. In this study, we cloned and characterized an atg12 homolog from orange spotted grouper (Epinephelus coioides) (Ecatg12). Ecatg12 encodes a 117 amino acid protein that shares 94.0% and 76.8% identity with gourami (Anabas_testudineus) and humans (Homo sapiens), respectively. The transcription level of Ecatg12 was lower in cells infected with Singapore grouper iridovirus (SGIV) than in non-infected cells. Fluorescence microscopy revealed that EcAtg12 localized in the cytoplasm and nucleus in grouper spleen cells. Overexpression of EcAtg12 significantly increased the replication of SGIV, as evidenced by increased severity of the cytopathic effect, transcription levels of viral genes, levels of viral proteins, and progeny virus yield. Further studies showed that EcAtg12 overexpression decreased the expression levels of interferon (IFN) related molecules and pro-inflammatory factors and inhibited the promoter activity of IFN-3, interferon-stimulated response element, and nuclear factor-κB. Together, these results demonstrate that EcAtg12 plays crucial roles in SGIV replication by downregulating antiviral immune responses.

Oyster Versatile IKKα/βs Are Involved in Toll-Like Receptor and RIG-I-Like Receptor Signaling for Innate Immune Response.

IκB kinases (IKKs) play critical roles in innate immunity through signal-induced activation of the key transcription factors nuclear factor-κB (NF-κB) and interferon regulatory factors (IRFs). However, studies of invertebrate IKK functions remain scarce. In this study, we performed phylogenetic analysis of IKKs and IKK-related kinases encoded in the Pacific oyster genome. We then cloned and characterized the oyster IKKα/β-2 gene. We found that oyster IKKα/β-2, a homolog of human IKKα/IKKβ, responded to challenge with lipopolysaccharide (LPS), peptidoglycan (PGN), and polyinosinic-polycytidylic acid [poly(I:C)]. As a versatile immune molecule, IKKα/β-2 activated the promoters of NF-κB, TNFα, and IFNβ, as well as IFN-stimulated response element (ISRE)-containing promoters, initiating an antibacterial or antiviral immune state in mammalian cells. Importantly, together with the cloned oyster IKKα/β-1, we investigated the signal transduction pathways mediated by these two IKKα/β proteins. Our results showed that IKKα/β-1 and IKKα/β-2 could interact with the oyster TNF receptor-associated factor 6 (TRAF6) and that IKKα/β-2 could also bind to the oyster myeloid differentiation factor 88 (MyD88) protein directly, suggesting that oyster IKKα/βs participate in both RIG-I-like receptor (RLR) and Toll-like receptor (TLR) signaling for the reception of upstream immune signals. The fact that IKKα/β-1 and IKKα/β-2 formed homodimers by interacting with themselves and heterodimers by interacting with each other, along with the fact that both oyster IKKα/β proteins interacted with NEMO protein, indicates that oyster IKKα/βs and the scaffold protein NEMO form an IKK complex, which may be a key step in phosphorylating IκB proteins and activating NF-κB. Moreover, we found that oyster IKKα/βs could interact with IRF8, and this may be related to the IKK-mediated activation of ISRE promotors and their involvement in the oyster “interferon (IFN)-like” antiviral pathway. Moreover, the expression of oyster IKKα/β-1 and IKKα/β-2 may induce the phosphorylation of IκB proteins to activate NF-κB. These results reveal the immune function of oyster IKKα/β-2 and establish the existence of mollusk TLR and RLR signaling mediated by IKKα/β proteins for the first time. Our findings should be helpful in deciphering the immune mechanisms of invertebrates and understanding the development of the vertebrate innate immunity network.

Metformin Inhibits the Type 1 IFN Response in Human CD4+ T Cells.

In systemic lupus erythematosus, defective clearance of apoptotic debris and activation of innate cells result in a chronically activated type 1 IFN response, which can be measured in PBMCs of most patients. Metformin, a widely used prescription drug for Type 2 diabetes, has a therapeutic effect in several mouse models of lupus through mechanisms involving inhibition of oxidative phosphorylation and a decrease in CD4+ T cell activation. In this study, we report that in CD4+ T cells from human healthy controls and human systemic lupus erythematosus patients, metformin inhibits the transcription of IFN-stimulated genes (ISGs) after IFN-α treatment. Accordingly, metformin inhibited the phosphorylation of pSTAT1 (Y701) and its binding to IFN-stimulated response elements that control ISG expression. These effects were independent of AMPK activation or mTORC1 inhibition but were replicated using inhibitors of the electron transport chain respiratory complexes I, III, and IV. This indicates that mitochondrial respiration is required for ISG expression in CD4+ T cells and provides a novel mechanism by which metformin may exert a therapeutic effect in autoimmune diseases.

The Nucleoprotein and Phosphoprotein of Peste des Petits Ruminants Virus Inhibit Interferons Signaling by Blocking the JAK-STAT Pathway

Peste des petits ruminants virus (PPRV) is associated with global peste des petits ruminants resulting in severe economic loss. Peste des petits ruminants virus dampens host interferon-based signaling pathways through multiple mechanisms. Previous studies deciphered the role of V and C in abrogating IFN-β production. Moreover, V protein directly interacted with signal transducers and activators of transcription 1 (STAT1) and STAT2 resulting in the impairment of host IFN responses. In our present study, PPRV infection inhibited both IFN-β- and IFN-γ-induced activation of IFN-stimulated response element (ISRE) and IFN-γ-activated site (GAS) element, respectively. Both N and P proteins, functioning as novel IFN response antagonists, markedly suppressed IFN-β-induced ISRE and IFN-γ-induced GAS promoter activation to impair downstream upregulation of various interferon-stimulated genes (ISGs) and prevent STAT1 nuclear translocation. Specifically, P protein interacted with STAT1 and subsequently inhibited STAT1 phosphorylation, whereas N protein neither interacted with STAT1 nor inhibited STAT1 phosphorylation as well as dimerization, suggesting that the N and P protein antagonistic effects were different. Though they differed in their relationship to STAT1, both proteins blocked JAK-STAT signaling, severely negating the host antiviral immune response. Our study revealed a new mechanism employed by PPRV to evade host innate immune response, providing a platform to study the interaction of paramyxoviruses and host response.

TRIM14 expression is regulated by IRF-1 and IRF-2.

Tripartite motif‐containing 14 (TRIM14) is a mitochondrial adaptor that promotes innate immune signaling and plays important roles in antiviral defense. Expression of TRIM14 is induced by interferon (IFN)‐I. However, the mechanism by which IFN‐I induces TRIM14 production is not yet determined. In this study, we have examined the function of TRIM14 promoter and found that a GC box and an IFN‐stimulated response element (ISRE) are necessary for the basal level transcription of TRIM14. We further observed that IFN‐I activates the TRIM14 promoter through the ISRE. In particular, interferon regulatory factor (IRF)‐1 and IRF‐2 bind to the TRIM14 promoter and activate transcription of TRIM14. Moreover, knockdown of IRF‐1 reduces the stimulation of TRIM14 transcription by IFN‐α, suggesting that IRF‐1 is involved in the activation of TRIM14 by IFN‐I. IRF‐2 has little effect on IFN‐α‐induced TRIM14 transcription but is essential for the basal transcription of TRIM14.

Effect of suboptimal temperature on the regulation of endogenous antigen presentation in a rainbow trout hypodermal fibroblast cell line

Rainbow trout (Oncorhynchus mykiss) face low environmental temperatures over winter months and during extreme low temperature events. Suboptimal temperatures are known to negatively impact the teleost immune system, although there is mixed evidence in rainbow trout as to the effect on the endogenous antigen processing and presentation pathway (EAPP). The EAPP is an important pathway for antiviral defense that involves the presentation of endogenous peptides on the cell surface for recognition by cytotoxic T cells. Using a rainbow trout hypodermal fibroblast (RTHDF) cell line as an in vitro model, we determined that constitutive EAPP transcript levels are not impaired at low temperature, but induction of up-regulation of these transcripts is delayed at the suboptimal temperature following exposure to poly(I:C) or viral haemorrhagic septicaemia virus IVb, which was still able to enter and replicate in the cell line at 4 °C, albeit with reduced efficiency. The delay in the induction of EAPP mRNA level up-regulation following poly(I:C) stimulation coincided with a delay in ifn1 transcript levels and secretion, which is important since interferon-stimulated response elements were identified in the promoter regions of the EAPP-specific members of the pathway, implying that IFN1 is involved in the regulation of these genes. Our results suggest that the ability of rainbow trout to mount an effective immune response to viral pathogens may be lessened at suboptimal temperatures.

Signal Integration of IFN-I and IFN-II With TLR4 Involves Sequential Recruitment of STAT1-Complexes and NFκB to Enhance Pro-inflammatory Transcription.

Atherosclerosis is a chronic inflammatory disease of the blood vessels, characterized by atherosclerotic lesion formation. Vascular Smooth Muscle Cells (VSMC), macrophages (MΦ), and dendritic cells (DC) play a crucial role in vascular inflammation and atherosclerosis. Interferon (IFN)α, IFNγ, and Toll-like receptor (TLR)4 activate pro-inflammatory gene expression and are pro-atherogenic. Gene expression regulation of many pro-inflammatory genes has shown to rely on Signal Integration (SI) between IFNs and TLR4 through combinatorial actions of the Signal Transducer and Activator of Transcription (STAT)1 complexes ISGF3 and γ-activated factor (GAF), and Nuclear Factor-κB (NFκB). Thus, IFN pre-treatment (“priming”) followed by LPS stimulation leads to enhanced transcriptional responses as compared to the individual stimuli. To characterize the mechanism of priming-induced IFNα + LPS- and IFNγ + LPS-dependent SI in vascular cells as compared to immune cells, we performed a comprehensive genome-wide analysis of mouse VSMC, MΦ, and DC in response to IFNα, IFNγ, and/or LPS. Thus, we identified IFNα + LPS or IFNγ + LPS induced genes commonly expressed in these cell types that bound STAT1 and p65 at comparable γ-activated sequence (GAS), Interferon-stimulated response element (ISRE), or NFκB sites in promoter proximal and distal regions. Comparison of the relatively high number of overlapping ISRE sites in these genes unraveled a novel role of ISGF3 and possibly STAT1/IRF9 in IFNγ responses. In addition, similar STAT1-p65 co-binding modes were detected for IFNα + LPS and IFNγ + LPS up-regulated genes, which involved recruitment of STAT1 complexes preceding p65 to closely located GAS/NFκB or ISRE/NFκB composite sites already upon IFNα or IFNγ treatment. This STAT1-p65 co-binding significantly increased after subsequent LPS exposure and correlated with histone acetylation, PolII recruitment, and amplified target gene transcription in a STAT1-p65 co-bound dependent manner. Thus, co-binding of STAT1-containing transcription factor complexes and NFκB, activated by IFN-I or IFN-II together with LPS, provides a platform for robust transcriptional activation of pro-inflammatory genes. Moreover, our data offer an explanation for the comparable effects of IFNα or IFNγ priming on TLR4-induced activation in vascular and immune cells, with important implications in atherosclerosis.

Inhibition of PARP1 Increases IRF-dependent Gene Transcription in Jurkat Cells.

Poly(ADP-ribose) polymerase 1 (PARP1) plays important roles in the regulation of transcription factors. Mounting evidence has shown that inhibition of PARP1 influences the expression of genes associated with inflammatory response. Interferon regulatory factor 1 (IRF1) is a critical transcription factor for the development of both the innate and adaptive immune responses against infections. However, the molecular mechanism through which PARP1 mediates the effects has not been clearly demonstrated. Jurkat cells were exposed to dexamethasone (Dex) or PARP1 inhibitor PJ34. The expression levels of IL-12, LMP2, OAS1 and PKR were detected using real-time RT-PCR. The interactions between PARP1 and IRF1 were examined by co-immunoprecipitation (co-IP) assays. We further explored the mechanism of PARP1 suppressing IRF1 by assessing the activities of interferon stimulated response element (ISRE). The mRNA expression of IL-12, LMP2, OAS1 and PKR was obviously suppressed by Dex in Jurkat cells, which could be rescued by PJ34 treatment. Luciferase study revealed that poly(ADP-ribosyl)- ation suppressed IRF1-mediated transcription through preventing the binding of IRF1 to ISREs. PARP1 inhibited IRF1-mediated transcription in Jurkat cells by preventing IRF1 binding to ISREs in the promoters of target genes. It is suggested that PARP1 is a crucial regulator of IRF1-mediated immune response. This study provides experimental evidence for the possible application of PARP1 inhibitors in the treatment of IRF1-related immune anergy.

Direct Inhibition of IRF-Dependent Transcriptional Regulatory Mechanisms Associated With Disease.

Interferon regulatory factors (IRFs) are a family of homologous proteins that regulate the transcription of interferons (IFNs) and IFN-induced gene expression. As such they are important modulating proteins in the Toll-like receptor (TLR) and IFN signaling pathways, which are vital elements of the innate immune system. IRFs have a multi-domain structure, with the N-terminal part acting as a DNA binding domain (DBD) that recognizes a DNA-binding motif similar to the IFN-stimulated response element (ISRE). The C-terminal part contains the IRF-association domain (IAD), with which they can self-associate, bind to IRF family members or interact with other transcription factors. This complex formation is crucial for DNA binding and the commencing of target-gene expression. IRFs bind DNA and exert their activating potential as homo or heterodimers with other IRFs. Moreover, they can form complexes (e.g., with Signal transducers and activators of transcription, STATs) and collaborate with other co-acting transcription factors such as Nuclear factor-κB (NF-κB) and PU.1. In time, more of these IRF co-activating mechanisms have been discovered, which may play a key role in the pathogenesis of many diseases, such as acute and chronic inflammation, autoimmune diseases, and cancer. Detailed knowledge of IRFs structure and activating mechanisms predisposes IRFs as potential targets for inhibition in therapeutic strategies connected to numerous immune system-originated diseases. Until now only indirect IRF modulation has been studied in terms of antiviral response regulation and cancer treatment, using mainly antisense oligonucleotides and siRNA knockdown strategies. However, none of these approaches so far entered clinical trials. Moreover, no direct IRF-inhibitory strategies have been reported. In this review, we summarize current knowledge of the different IRF-mediated transcriptional regulatory mechanisms and how they reflect the diverse functions of IRFs in homeostasis and in TLR and IFN signaling. Moreover, we present IRFs as promising inhibitory targets and propose a novel direct IRF-modulating strategy employing a pipeline approach that combines comparative in silico docking to the IRF-DBD with in vitro validation of IRF inhibition. We hypothesize that our methodology will enable the efficient identification of IRF-specific and pan-IRF inhibitors that can be used for the treatment of IRF-dependent disorders and malignancies.

Ononin, sec-O-β-d-glucosylhamaudol and astragaloside I: antiviral lead compounds identified via high throughput screening and biological validation from traditional Chinese medicine Zhongjing formulary

Traditional medicine (TM) is a valuable source for drug discovery. The knowledge in healing traditions has led to the success of some of the best-known drugs. However, the concept of ancient medical knowledge, such as herbal remedies and their therapeutic experience is rarely used in the current methodologies for developing therapeutics from TM. As a result, the screening procedure of TM compounds remains tough and labor-intensive. This study aimed to develop a new strategy that is capable of efficiently identifying antiviral leads from complex traditional Chinese medicine (TCM) by integrating knowledge from ancient healing practices with luciferase-based high-throughput screening (HTS). ‘Shanghan Zabing Lun’, an ancient TCM treatise which contains over 200 formulae was selected for knowledge mining based on its antiviral activity. Thirty formulae were rationally selected and utilized for the preparation of a 1306-fraction herbal formulae extract library by standardized chromatographic fractionation. The antiviral activity of the library was screened on a HEK293T cell model carrying a luciferase-driven interferon stimulated response element (ISRE). Hit compounds were further identified using liquid chromatography mass spectrometry analysis, and the mechanism of action of which were preliminarily explored through western blotting and immunofluorescence. A total of 18 fractions and 3 compounds were found activating ISRE. The three compounds, namely ononin, sec-O-β-d-glucosylhamaudol and astragaloside I could activate p65 phosphorylation and nuclear translocation. By doing so, a new strategy termed Knowledge-Based High Throughput Screening (KB-HTS) has been established, which provides an alternative approach to unearth antiviral lead compounds from TM. This new discovery pipeline can expedite the discovery process by improving dereplication and lead prioritization strategies, which is valuable for novel lead discovery from traditional medicine.

PU.1 and IRF8 regulate the transcriptional landscapes differently in naïve and activated B cells

Abstract The Ets family member PU.1 and the IRF family member IRF8 play crucial roles in regulating hematopoietic cell development including B cells. Both factors are constitutively expressed at high levels in B cells and control plasma cell differentiation. IRF8 and PU.1 function either together as a heterodimer or by pairing with other factors to bind target DNA sequences. Several consensus binding sequences for IRF8 and PU.1 have been identified, including the canonical interferon stimulated response element (ISRE) sequence motif (GAAANNGAAA), the Ets-IRF composite element (EICE) (GGAANNGAAA) and the IRF-Ets composite sequence (IECS) (GAAANN[N]GGAA). Although the target genes of IRF8 and PU.1 have been studied previously by chromatin immunoprecipitation (ChIP), the nature and the importance of the motifs engaging both PU.1 and IRF8 in B cells have not been determined. In this study, we performed ChIP-seq using sort-purified naïve follicular B cells from C57BL/6 mice that were either not stimulated or stimulated with anti-IgM plus anti-CD40, which mimic signals for germinal center B cell differentiation. We also integrated our ChIP-seq data with RNA-seq data from B cells deficient for both IRF8 and PU.1, and found that IRF8 and PU.1 regulate gene expression by preferentially binding to the EICE motifs of target genes known to maintain follicular B cell identity, localization, and survival. In contrast, in activated B cells, the binding landscape of IRF8/PU.1 in target genes shifts to IRF8-dominant ISRE motifs, possibly due to elevated expression of IRF8. These results provide new insights into our understanding of the molecular mechanisms underlying the transcriptional control of late stage B cell development.

Maternal recognition of pregnancy

Enhanced secretion of PGF2 alpha from endometrial explants in vitro in response to oxytocin is associated with augmented activities of phospholipase A2, phospholipase C and prostaglandin endoperoxide H synthase (PGS). In early pregnancy, maintenance of the corpus luteum is associated with an absence of pulsatile PGF2 alpha secretion; an increase in endometrial inhibitors of phospholipase A2 and PGS contribute to the antiluteolytic alterations of PGF2 alpha secretion. Linoleic acid is a competitive inhibitor of arachidonic acid metabolism by PGS, and microsomal concentrations of free linoleic acid are increased in the endometrium of pregnant cattle. The trophoblast produces large quantities of interferon tau (IFN-tau). Inhibition of increases in endometrial oestradiol receptor mRNA and protein are associated with intrauterine administration of recombinant (r) ovine (o) IFN-tau in sheep. Intrauterine injections of ovine (b) IFN-tau in cattle (days 14-17) altered endometrial function so that secretion of PGF2 alpha from cultured endometrial epithelial cells was reduced. Antiluteolytic effects were not expressed in 20% of cows receiving IFN-tau or rbIFN-alpha I1 indicating that an inadequate endometrial responsiveness may contribute to embryo mortality. IFN-tau may activate a signal transduction system similar to that induced by other type I IFNs; activation of an intracellular tyrosine kinase ultimately leads to activation of an IFN-stimulated response element to induce gene transcription. Biological responses associated with pregnancy and IFN-tau treatment are integrated into a multifactorial antiluteolytic model. Strategies to enhance embryo survival could include supplementation with rIFN-tau and alterations in endometrial responsiveness to this cytokine through dietary manipulation of lipid metabolism.

Activation of interferon-α signaling by resveratrol, genistein and quercetin

Resveratrol, genistein and quercetin from the group of polyphenols from secondary plant metabolites reveal cancer preventive and antivirus effects realized via their pleiotropic influence on the different macromolecules in cells. These compounds can interact with DNA without the formation of covalent bonds. This process is usually followed by changes in spatial, physical-chemical and structural DNA characteristics that can result in disfunction of DNA metabolism enzymes and chromatin destabilization. Similar effects were described for anticancer drug Curaxine CBL0137 in association with activation of interferon-α signaling. We demonstrated dose-dependent stimulating effects of resveratrol, genistein and quercetin on interferon-α signaling using HeLa cells expressed mCherry protein with interferon-stimulated response elements (ISRE) in promoter. Furthermore, it was shown by live-cell fluorescent microscopy in HT1080 cells with mCherry-labeled histone H1.5 that described polyphenols induced the redistribution of this linker histone in cell nuclei. The data obtained suggest an existence of DNA-dependent mechanism of anticancer effects of plant polyphenols and a need for further study of crosslinks between the polyphenols’ influence on chromatin structure and the changes in genome function, in particular, induction of interferon- interferon-α signaling.