Regulation Of Interferon-stimulated Genes Research Articles

SCD2-mediated cooperative activation of IRF3-IRF9 regulatory circuit controls type I interferon transcriptome in CD4+ T cells

Type I interferons (type I-IFN) are critical for the host defense to viral infection, and at the same time, the dysregulation of type I-IFN responses leads to autoinflammation or autoimmunity. Recently, we reported that the decrease in monounsaturated fatty acid caused by the genetic deletion of Scd2 is essential for the activation of type I-IFN signaling in CD4+ Th1 cells. Although interferon regulatory factor (IRF) is a family of homologous proteins that control the transcription of type I-IFN and interferon stimulated genes (ISGs), the member of the IRF family that is responsible for the type I-IFN responses induced by targeting of SCD2 remains unclear. Here, we report that the deletion of Scd2 triggered IRF3 activation for type I-IFN production, resulting in the nuclear translocation of IRF9 to induce ISG transcriptome in Th1 cells. These data led us to hypothesize that IRF9 plays an essential role in the transcriptional regulation of ISGs in Scd2-deleted (sgScd2) Th1 cells. By employing ChIP-seq analyses, we found a substantial percentage of the IRF9 target genes were shared by sgScd2 and IFNβ-treated Th1 cells. Importantly, our detailed analyses identify a unique feature of IRF9 binding in sgScd2 Th1 cells that were not observed in IFNβ-treated Th1 cells. In addition, our combined analyses of transcriptome and IRF9 ChIP-seq revealed that the autoimmunity related genes, which increase in patient with SLE, were selectively increased in sgScd2 Th1 cells. Thus, our findings provide novel mechanistic insights into the process of fatty acid metabolism that is essential for the type I-IFN response and the activation of the IRF family in CD4+ T cells.

Transient Increases in Inflammation and Proapoptotic Potential Are Associated with the HESN Phenotype Observed in a Subgroup of Kenyan Female Sex Workers.

Interferon (IFN) -stimulated genes (ISGs) are critical effectors of IFN response to viral infection, but whether ISG expression is a correlate of protection against HIV infection remains elusive. A well-characterized subcohort of Kenyan female sex workers, who, despite being repeatedly exposed to HIV-1 remain seronegative (HESN), exhibit reduced baseline systemic and mucosal immune activation. This study tested the hypothesis that regulation of ISGs in the cells of HESN potentiates a robust antiviral response against HIV. Transcriptional profile of a panel of ISGs with antiviral function in PBMC and isolated CD4+ T cells from HESN and non-HESN sex worker controls were defined following exogenous IFN-stimulation using relative RT-qPCR. This study identified a unique profile of proinflammatory and proapoptotic ISGs with robust but transient responses to exogenous IFN-γ and IFN-α2 in HESN cells. In contrast, the non-HESN cells had a strong and prolonged proinflammatory ISG profile at baseline and following IFN challenge. Potential mechanisms may include augmented bystander apoptosis due to increased TRAIL expression (16-fold), in non-HESN cells. The study also identified two negative regulators of ISG induction associated with the HESN phenotype. Robust upregulation of SOCS-1 and IRF-1, in addition to HDM2, could contribute to the strict regulation of proinflammatory and proapoptotic ISGs in HESN cells. As reducing IRF-1 in the non-HESN cells resulted in the identified HESN ISG profile, and decreased HIV susceptibility, the unique HESN ISG profile could be a correlate of protection against HIV infection.

Regulation of interferon stimulated gene expression levels at homeostasis

Interferon stimulated genes (ISGs), a collection of genes important in the early innate immune response, are upregulated in response to stimulation by extracellular type I interferons. The regulation of ISGs has been extensively studied in cells exposed to significant interferon stimulation, but less is known about ISG regulation in homeostatic regimes in which extracellular interferon levels are low. Using a collection of pre-existing, publicly available microarray datasets, we investigated ISG regulation at homeostasis in CD4, pulmonary epithelial, fibroblast and macrophage cells. We used a linear regression model to predict ISG expression levels from regulator expression levels. Our results suggest significant regulation of ISG expression at homeostasis, both through the ISGF3 molecule and through IRF7 and IRF8 associated pathways. We find that roughly 50% of ISGs have expression levels significantly correlated with ISGF3 expression levels at homeostasis, supporting previous results suggesting that homeostatic IFN levels have broad functional consequences. We find that ISG expression levels varied in their correlation with ISGF3, with epithelial and macrophage cells showing more correlation than CD4 and fibroblast cells. Our analysis provides a novel approach for decomposing and quantifying ISG regulation.

Interferon-Stimulated Genes Are Transcriptionally Repressed by PR in Breast Cancer.

The progesterone receptor (PR) regulates transcriptional programs that drive proliferation, survival, and stem cell phenotypes. Although the role of native progesterone in the development of breast cancer remains controversial, PR clearly alters the transcriptome in breast tumors. This study identifies a class of genes, Interferon (IFN)-stimulated genes (ISGs), potently downregulated by ligand-activated PR which have not been previously shown to be regulated by PR. Progestin-dependent transcriptional repression of ISGs was observed in breast cancer cell line models and human breast tumors. Ligand-independent regulation of ISGs was also observed, as basal transcript levels were markedly higher in cells with PR knockdown. PR repressed ISG transcription in response to IFN treatment, the canonical mechanism through which these genes are activated. Liganded PR is robustly recruited to enhancer regions of ISGs, and ISG transcriptional repression is dependent upon PR's ability to bind DNA. In response to PR activation, key regulatory transcription factors that are required for IFN-activated ISG transcription, STAT2 and IRF9, exhibit impaired recruitment to ISG promoter regions, correlating with PR/ligand-dependent ISG transcriptional repression. IFN activation is a critical early step in nascent tumor recognition and destruction through immunosurveillance. As the large majority of breast tumors are PR positive at the time of diagnosis, PR-dependent downregulation of IFN signaling may be a mechanism through which early PR-positive breast tumors evade the immune system and develop into clinically relevant tumors.Implications: This study highlights a novel transcriptional mechanism through which PR drives breast cancer development and potentially evades the immune system. Mol Cancer Res; 15(10); 1331-40. ©2017 AACR.

Maternal recognition of pregnancy and implantation are not associated with an interferon response of the endometrium to the presence of the conceptus in dromedary camel

Maternal recognition of pregnancy (MRP) and implantation involve appropriate interactions between the elongating conceptus and the receptive endometrium that will condition development of the feto-placental unit to term. Molecular mechanisms that take place at the conceptus-endometrium interface during early pregnancy have been extensively investigated in domestic ungulates but they are still poorly understood in camelids including the dromedary camel (Camelus dromedarius), a domestic species with important economic and social roles in arid and semi-arid areas. In order to better understand how MRP and implantation take place in the left horn of this species, we investigated expression levels of genes encoding steroid hormones (PGR, ESR1), transcription factors (STAT1, FOXL2), interferon stimulated genes (MX1, MX2, OAS1, RSAD2) including SOCS genes (SOCS1, SOCS2, SOCS3 and CISH), previously identified as conceptus regulated genes in the endometrium of other domestic animals. Using endometrial tissue collected from left and right uterine horns of dromedary camel females that were non pregnant or early pregnant, gene expression of these genes was detected and our results provided first insights on their regulation, showing that (i) conceptus implantation is not associated with an IFN response in the pregnant uterine horn (ii) when regulation of classical interferon-stimulated genes (ISG) occurs, it takes place during the formation of the feto-placental unit, and (iii) gene expression can differ between the left and right uterine horns during implantation and early placentation phase. Additional experiments will be required in dromedary camels to understand the unusual regulation of ISG during implantation as well as to determine the molecular processes that drive the systematic implantation of the elongating conceptus in the left uterine horn.

Abstract 717: Overexpression of interferon-stimulated genes is critical for the survival of aromatase inhibitor-resistant breast cancer cells

Abstract Aromatase inhibitors (AIs) successfully treat many estrogen receptor expressing breast cancers by depleting circulating estrogen levels. Unfortunately, the majority of patients treated with AIs eventually develop resistance to this treatment. In order to elucidate the mechanism(s) of AI-resistance our lab has developed an AI-resistant breast cancer cell line MCF-7:5C which was clonally derived from parental MCF-7 cells following long-term estrogen deprivation. The AI-resistant MCF-7:5C cells grow robustly in the absence of estrogen, are highly aggressive, and constitutively overexpress a panel of type 1 interferon (IFN) stimulated genes (ISGs) including IFITM1, IFIT1, IFI27, PLSCR1, STAT1/2, OAS1 and MX1 as compared to the AI-sensitive MCF-7 cells. Human tissue microarray analysis also indicates that IFITM1 and PLSCR1 are overexpressed in AI-resistant/recurrence breast tumors as compared to primary tumors. ISGs are not expressed basally in normal tissue and are only induced by type1 IFNs (IFNα and β) to protect the host from viral infections. IFNα signals through a specific receptor, IFNAR1/2, which uses JAK/STAT signaling to produce the ISGs. The significance of constitutive overexpression of ISGs in AI-resistant breast cancer is not known. In this study, we investigated the functional importance of ISGs in the AI-resistant breast cancer cell line, MCF-7:5C, and the mechanism(s) that drive their overexpression in these cells. Using ELISA, we detected a 3-fold higher level of IFNα in AI-resistant MCF-7:5C cells than in the parental MCF-7 cells and FACS analysis revealed that the IFNα was produced by all cells in culture, not a distinct subset of the population. Blockade of IFNα signaling using a neutralizing antibody to IFNAR, as well as transient knockdown of IFNα and its transcription factor IRF7 dramatically reduced ISG expression. Most notably, loss of IFNα and suppression of type 1 IFN signaling resulted in the death of the AI-resistant MCF-7:5C cells, while having no effect on the parental MCF-7 cells, thus confirming its pro-survival function. Apart from IFNα signaling, we also found evidence of post-transcriptional regulation of ISGs in the AI-resistant cells. Specifically, several of the ISGs had a significantly longer mRNA half-life in the MCF-7:5C cells than in the parental MCF-7 cells. Additionally, knockdown of the RNA binding protein Human Antigen R (HuR) significantly reduced the mRNA stability and expression of the ISGs. Electron microscopy confirmed that AI-resistant MCF-7:5C cells exhibit classic morphological signs of anti-viral response and ISG expression, including dilated ER, and enhanced cell-cell adhesion as compared with the AI-sensitive MCF-7 cell line. Overall, we have found that AI-resistant breast cancer cells maintain elevated levels of IFNα in order to drive constitutive overexpression of ISGs which sustains their survival in estrogen deprived conditions. Citation Format: Asona Lui, Hye-Joung Choi, Joan Lewis-Wambi. Overexpression of interferon-stimulated genes is critical for the survival of aromatase inhibitor-resistant breast cancer cells. [abstract]. In: Proceedings of the 106th Annual Meeting of the American Association for Cancer Research; 2015 Apr 18-22; Philadelphia, PA. Philadelphia (PA): AACR; Cancer Res 2015;75(15 Suppl):Abstract nr 717. doi:10.1158/1538-7445.AM2015-717

Assessment of mTOR-Dependent Translational Regulation of Interferon Stimulated Genes.

Type-I interferon (IFN)-induced activation of the mammalian target of rapamycin (mTOR) signaling pathway has been implicated in translational control of mRNAs encoding interferon-stimulated genes (ISGs). However, mTOR-sensitive translatomes commonly include mRNAs with a 5’ terminal oligopyrimidine tract (TOP), such as those encoding ribosomal proteins, but not ISGs. Because these translatomes were obtained under conditions when ISG expression is not induced, we examined the mTOR-sensitive translatome in human WISH cells stimulated with IFN β. The mTOR inhibitor Torin1 resulted in a repression of global protein synthesis, including that of ISG products, and translation of all but 3 ISG mRNAs (TLR3, NT5C3A, and RNF19B) was not selectively more sensitive to mTOR inhibition. Detailed studies of NT5C3A revealed an IFN-induced change in transcription start site resulting in a switch from a non-TOP to a TOP-like transcript variant and mTOR sensitive translation. Thus, we show that, in the cell model used, translation of the vast majority of ISG mRNAs is not selectively sensitive to mTOR activity and describe an uncharacterized mechanism wherein the 5’-UTR of an mRNA is altered in response to a cytokine, resulting in a shift from mTOR-insensitive to mTOR-sensitive translation.

The role of IFI16 in the innate immune response to cytosolic nucleic acids (P1395)

Abstract Recently, the Interferon Gamma Inducible (IFI)16 was linked to cytoplasmic DNA induced type I interferon (IFN) responses. IFI16 was shown to associate with Stimulator of IFN genes (STING), leading to TANK binding kinase-1 (TBK1) dependent phosphorylation of interferon regulatory factor 3 (IRF3) and transcription of type I IFN genes. Here we created THP1 monocytic cell lines with stable knockdown of IFI16 by lentiviral transduction of shRNA and demonstrated that IFI16 knockdown leads to a severely attenuated type I IFN response following treatment of cells with either synthetic DNA ligands, or after infection with DNA viruses such as HSV-1. Surprisingly, these cells were also compromised in their ability to induce type I IFNs to other non-DNA ligands including synthetic 5’pppRNA and Sendai Virus, both of which signal via RIG-I. In contrast the NF-kB regulated cytokines IL-6 and IL-1β remain unaffected in IFI16 knockdown cells. Utilizing Nanostring technology, we also determined that IFI16 is capable of regulating DNA and RNA induced expression of a panel of Interferon Stimulated genes (ISGs), including RIG-I. We also monitored key steps in the type 1 IFN pathway and found that levels of total IRF3 were also reduced in IFI16 knockdown cells. These results suggest that IFI16 senses DNA viruses, eliciting a type I IFN response, but also acts more broadly in the regulation of ISGs in response to both DNA and RNA viruses.

IFN-Independent Expression of RIG-I is a Determinant of Heterogeneous IFN-B Expression States in Antiviral Signaling

RIG-I plays a major role in antiviral innate immunity by detecting the cytoplasmic viral RNA and triggering the pathway that leads to transcriptional activation of type I interferon (IFN). Type I IFN induces a large set of genes called interferon stimulated genes (ISGs) which coordinate to antagonize viral evasion. We utilized single-molecule fluorescence in situ hybridization (smFISH) to study viral and antiviral gene expression in individual cells. We precisely quantified kinetics of mRNA expression of RIG-I, IFN-b and Sendai virus (SeV) L gene in clonal mammalian cells upon infection with SeV. Contrary to the expectation that RIG-I expression is only up-regulated through positive feedback involving Type I IFN, we found that RIG-I is directly induced by viral infection in the absence of IFN at the early stages of viral infection. Examination of expression kinetics of a subset of ISGs showed the differential regulation of ISGs in response viral infection. MDA5, LGP2, OasL and Viperin were directly activated by SeV infection before IFN-b. On the other hand, activation of some other ISGs was found to be dependent on IFN signaling, such as PKR, MxA, IRF7, TRIM25, and NLRX1. smFISH experiments revealed two distinct populations of cells which would otherwise be obscured in ensemble measurements: IFN-b expressing and IFN-b non-expressing cells. Simultaneous counting of IFN-b, RIG-I and SeV L gene mRNA and correlation analysis in individual cells revealed that transcriptional activation of IFN-b is dependent on the level of RIG-I mRNA in individual cells that is IFN-independently induced at the early stages of viral infection but is independent of the level of viral replication.

O039 The interferonscape: Systems biology of interferon stimulated pathways and networks

Introduction The Interferons (IFNs) are a family of pleiotropic cytokines that mediate anti-microbial, anti-proliferative, anti-tumour, immuno-modulatory and homoeostatic host defence functions. Differential regulation of Interferon Stimulated Genes (ISGs) by IFNs, via multiple signalling pathways and the combinatorial effects of IFN-induced transcription factors, co-regulators, ncRNA and epigenetic modifications underlie these diverse biological functions. Methods We have built InterferonScape – a software platform for data-integration, mining and visualization to study IFN mediated ISG regulation at a systems level. This is a centralized systems-immunology resource that integrates ISG datasets into a data warehouse with a multidimensional data analysis capability. Statistical and computational data-mining methods combined with easy to use graphical user interfaces are included to provide custom-built data-visualization capability. This resource is provided as a web 2.0 rich internet application with novel embedded data-mining, search and semantic web technologies. Results Using integrative data-mining approaches we previously identified approximately 1800 ISGs in the human genome [1] . Very few of these ISGs have been studied systematically, and information on ISG pathways is virtually absent from both public and commercial pathway repositories. We have utilised computational pathway modelling approaches, coupled to natural language text mining and expert manual curation to build a reliable collection of ISG pathways and networks. By integrating both in-house generated and publicly-available gene expression, regulatory genomic and epigenetic datasets in different cell and tissue types and in both normal and disease conditions, we have identified the regulatory elements and promoter modules involved in the regulation of ISG networks. Conclusion Using integrative genomic and computational biology approaches we will demonstrate the evolutionary conservation, information flow, connectivity, network topology and regulatory interactions underlying these ISG networks. Understanding the propagation of ISG networks in different conditions will provide a greater insight into IFN biology in health and disease.

PS2-75 Regulation of the response to interferon therapy through innate immune tolerance during hepatitis C virus infection

Maternal recognition of pregnancy (MRP) and implantation involve appropriate interactions between the elongating conceptus and the receptive endometrium that will condition development of the feto-placental unit to term. Molecular mechanisms that take place at the conceptus-endometrium interface during early pregnancy have been extensively investigated in domestic ungulates but they are still poorly understood in camelids including the dromedary camel (Camelus dromedarius), a domestic species with important economic and social roles in arid and semi-arid areas. In order to better understand how MRP and implantation take place in the left horn of this species, we investigated expression levels of genes encoding steroid hormones (PGR, ESR1), transcription factors (STAT1, FOXL2), interferon stimulated genes (MX1, MX2, OAS1, RSAD2) including SOCS genes (SOCS1, SOCS2, SOCS3 and CISH), previously identified as conceptus regulated genes in the endometrium of other domestic animals. Using endometrial tissue collected from left and right uterine horns of dromedary camel females that were non pregnant or early pregnant, gene expression of these genes was detected and our results provided first insights on their regulation, showing that (i) conceptus implantation is not associated with an IFN response in the pregnant uterine horn (ii) when regulation of classical interferon-stimulated genes (ISG) occurs, it takes place during the formation of the feto-placental unit, and (iii) gene expression can differ between the left and right uterine horns during implantation and early placentation phase. Additional experiments will be required in dromedary camels to understand the unusual regulation of ISG during implantation as well as to determine the molecular processes that drive the systematic implantation of the elongating conceptus in the left uterine horn.

Regulation of Interferon-Stimulated Genes in Peripheral Blood Leukocytes in Pregnant and Bred, Nonpregnant Dairy Cows

In ruminants, pregnancy results in up-regulation of a large number of IFN-stimulated genes (ISG) in the uterus. Recently, one of these genes was also shown to increase in peripheral blood leukocytes (PBL) during early pregnancy in sheep. Our working hypothesis is that conceptus signaling activates maternal gene expression in PBL in dairy cattle. The objectives of this study were to characterize ISG expression in PBL from pregnant (n=20) and bred, nonpregnant (n=30) dairy cows. Steady-state levels of mRNA for Mx1, Mx2, β2-microglobulin, ISG-15, IFN regulatory factor-1, and IFN regulatory factor-2 were quantified. Holstein cows were synchronized to estrus and artificially inseminated (d 0). Blood samples were collected (coccygeal venipuncture) on d 0 and 16, 18, and 20 d after insemination for progesterone analysis and PBL isolation. Pregnancy was confirmed by transrectal ultrasonography at approximately 40 d after breeding. A status×day interaction was detected for Mx1, Mx2, and ISG-15 gene expression. When analyzed within day, levels of mRNA for ISG-15 and Mx1 were greater in pregnant compared with bred, nonpregnant cows on d 18 and 20, respectively. Expression of the Mx2 gene increased in the pregnant group compared with bred, nonpregnant cows on d 16, 18, and 20 after insemination. β2-Microglobulin, IFN regulatory factor-1, and IFN regulatory factor-2 were not different between groups. The results clearly indicated that components of the innate immune response are activated in PBL during the period of pregnancy recognition and early embryo signaling. The physiological implications of these changes on maternal immune function are as yet unknown; however, they do provide a unique opportunity to identify bred, nonpregnant, cows 18 d after insemination in dairy cattle.

Transcriptional profiling of interferon regulatory factor 3 target genes: direct involvement in the regulation of interferon-stimulated genes.

Ubiquitously expressed interferon regulatory factor 3 (IRF-3) is directly activated after virus infection and functions as a key activator of the immediate-early alpha/beta interferon (IFN) genes, as well as the RANTES chemokine gene. In the present study, a tetracycline-inducible expression system expressing a constitutively active form of IRF-3 (IRF-3 5D) was combined with DNA microarray analysis to identify target genes regulated by IRF-3. Changes in mRNA expression profiles of 8,556 genes were monitored after Tet-inducible expression of IRF-3 5D. Among the genes upregulated by IRF-3 were transcripts for several known IFN-stimulated genes (ISGs). Subsequent analysis revealed that IRF-3 directly induced the expression of ISG56 in an IFN-independent manner through the IFN-stimulated responsive elements (ISREs) of the ISG56 promoter. These results demonstrate that, in addition to its role in the formation of a functional immediate-early IFN-beta enhanceosome, IRF-3 is able to discriminate among ISRE-containing genes involved in the establishment of the antiviral state as a direct response to virus infection.