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The key role of structural cells in immune modulation has been revealed with the advent of single-cell multiomics, but the underlying mechanism remains poorly understood. Here, we revealed that the transcriptional activation of interferon regulatory factor 1 (IRF1) in response to ionizing radiation, cytotoxic chemicals and SARS-CoV-2 viral infection determines the fate of structural cells and regulates communication between structural and immune cells. Radiation-induced leakage of mtDNA initiates the nuclear translocation of IRF1, enabling it to regulate the transcription of inflammation- and cell death-related genes. Novel posttranslational modification (PTM) sites in the nuclear localization sequence (NLS) of IRF1 were identified. Functional analysis revealed that mutation of the acetylation site and the phosphorylation sites in the NLS blocked the transcriptional activation of IRF1 and reduced cell death in response to ionizing radiation. Mechanistically, reciprocal regulation between the single-stranded DNA sensors SSBP1 and IRF1, which restrains radiation-induced and STING/p300-mediated PTMs of IRF1, was revealed. In addition, genetic deletion or pharmacological inhibition of IRF1 tempered radiation-induced inflammatory cell death, and radiation mitigators also suppressed SARS-CoV-2 NSP-10-mediated activation of IRF1. Thus, we revealed a novel cytoplasm-oriented mechanism of IRF1 activation in structural cells that promotes inflammation and highlighted the potential effectiveness of IRF1 inhibitors against immune disorders.

Background Interferon regulatory factor 1 (IRF1) is an important transcription factor that activates the type I interferon (IFN-I) response and plays a vital role in the antiviral immune response. Although IRF1 has been identified in several mammals, little information related to its function in canines has been described. Results In this study, canine IRF1 (CaIRF1) was cloned. After a series of bioinformatics analyses, we found that the CaIRF1 protein structure was similar to that of other animal IRF1 proteins, including a conserved DNA-binding domain (DBD), an IRF-association domain 2 (IAD2) domain and two nuclear localization signals (NLSs). An indirect immunofluorescence assay (IFA) revealed that CaIRF1 was mainly distributed in the nucleus. Overexpression of CaIRF1 in Madin-Darby canine kidney cells (MDCK) induced high levels of interferon β (IFNβ) and IFN-stimulated response element (ISRE) promoter activation and induced interferon-stimulated gene (ISG) expression. Subsequently, we assayed the antiviral activity of CaIRF1 against vesicular stomatitis virus (VSV) and canine parvovirus type-2 (CPV-2) in MDCK cells. Overexpression of CaIRF1 effectively inhibited the viral yields of VSV and CPV-2, while knocking down of CaIRF1 expression mildly increased viral gene copies. Conclusions CaIRF1 is involved in the cellular IFN-I signaling pathway and plays an important role in the antiviral response.

Classical swine fever (CSF) is caused by the classical swine fever virus (CSFV), which poses a threat to swine production. The activation of host innate immunity through linker proteins such as tumor necrosis factor receptor (TNF-R)-associated factor (TRAF) is crucial for the induction of the NF-κB pathway. Recent research has revealed the involvement of mitochondrial antiviral-signaling protein (MAVS) in the interaction with TRAF2, 3, 5, and 6 to activate both the NF-κB and IRF3 pathways. This study revealed that CSFV infection led to the upregulation of TRAF1 mRNA and protein levels; moreover, TRAF1 overexpression inhibited CSFV replication, while TRAF1 knockdown promoted replication, highlighting its importance in the host response to CSFV infection. Additionally, the expression of RIG-I, MAVS, TRAF1, IRF1, and ISG15 were detected in PK-15 cells infected with CSFV, revealing that TRAF1 plays a role in regulating IRF1 and ISG15 within the RIG-I pathway. Furthermore, Co-IP, GST pull-down, and IFA analyses demonstrated that TRAF1 interacted with MAVS and co-localized in the cytoplasm during CSFV infection. Ultimately, TRAF1 acted as a novel member of the TRAF family, bound to MAVS as a linker molecule, and functioned as a mediator downstream of MAVS in the RIG-I/MAVS pathway against CSFV replication.

Viral infections have been proposed to elicit pathological processes leading to the initiation of T helper 1 (TH1) immunity against dietary gluten and celiac disease (CeD). To test this hypothesis and gain insights into mechanisms underlying virus-induced loss of tolerance to dietary antigens, we developed a viral infection model that makes use of two reovirus strains that infect the intestine but differ in their immunopathological outcomes. Reovirus is an avirulent pathogen that elicits protective immunity, but we discovered that it can nonetheless disrupt intestinal immune homeostasis at inductive and effector sites of oral tolerance by suppressing peripheral regulatory T cell (pTreg) conversion and promoting TH1 immunity to dietary antigen. Initiation of TH1 immunity to dietary antigen was dependent on interferon regulatory factor 1 and dissociated from suppression of pTreg conversion, which was mediated by type-1 interferon. Last, our study in humans supports a role for infection with reovirus, a seemingly innocuous virus, in triggering the development of CeD.

Interleukin-10 (IL-10) is pivotal in suppressing innate immune activation, in large part by suppressing induction of inflammatory genes. Despite decades of research, the molecular mechanisms underlying this inhibition have not been resolved. Here we utilized an integrated epigenomic analysis to investigate IL-10-mediated suppression of LPS and TNF responses in primary human monocytes. Instead of inhibiting core TLR4-activated pathways such as NF-κB, MAPK–AP-1 and TBK1–IRF3 signaling, IL-10 targeted IRF transcription factor activity and DNA binding, particularly IRF5 and an IRF1-mediated amplification loop. This resulted in suppression of inflammatory NF-κB target genes and near-complete suppression of interferon-stimulated genes. Mechanisms of gene inhibition included downregulation of chromatin accessibility, de novo enhancer formation and IRF1-associated H3K27ac activating histone marks. These results provide a mechanism by which IL-10 suppresses inflammatory NF-κB target genes, highlight the role of IRF1 in inflammatory gene expression and describe the suppression of IFN responses by epigenetic mechanisms. Ivashkiv and colleagues show that IL-10 inhibits the expression and DNA binding of IRF1 and IRF5, two transcription factors that have an amplifying role in the induction of inflammatory NF-κB target genes and directly induce the expression of ISGs.

The eponymous member of the interferon regulatory factor (IRF) family, IRF1, was originally identified as a nuclear factor that binds and activates the promoters of type I interferon genes. However, subsequent studies using genetic knockouts or RNAi-mediated depletion of IRF1 provide a much broader view, linking IRF1 to a wide range of functions in protection against invading pathogens. Conserved throughout vertebrate evolution, IRF1 has been shown in recent years to mediate constitutive as well as inducible host defenses against a variety of viruses. Fine-tuning of these ancient IRF1-mediated host defenses, and countering strategies by pathogens to disarm IRF1, play crucial roles in pathogenesis and determining the outcome of infection.

Interferon regulatory factors (IRFs) play key roles during viral and bacterial infections. However, their regulation by inflammatory cytokines, including interleukin (IL)-1β and tumor necrosis factor (TNF) α, remains underexplored. As airway epithelial cells (AECs) modulate lung inflammation, IRF expression was characterized in pulmonary A549 and bronchial BEAS-2B epithelial cells along with primary AECs grown in submersion, or air-liquid interface, culture. While, IRF6 mRNA was only highly expressed in primary cells, IRF4 and IRF8 mRNAs were consistently low across the models. All the other IRF mRNAs were expressed in each model. IRF3 and IRF9 mRNAs were highly expressed, but their proteins remained primarily cytoplasmic post-IL-1β treatment in A549 cells. IRF2 showed moderate/high mRNA expression and was constitutively nuclear. However, RNA silencing did not support roles for IRF2 or IRF3, with only a modest role for IRF9, in the IL-1β-induced activation of an IRF reporter. IRF1 mRNA was highly induced by IL-1β in A549 and primary cells. Similarly, IRF1 protein was increased by IL-1β and TNFα in A549 cells, and by TNFα in BEAS-2B cells. In A549 cells, IL-1β-induced IRF1 protein localized to the nucleus and since IRF1 silencing prevented IRF reporter activity, a major transcriptional role was indicated. Mechanistically, the inflammatory transcription factor, nuclear factor (NF)-κB, was necessary for IL-1β- and TNFα-induced IRF1 expression. Further, four novel enhancer regions 5' to IRF1 bound the NF-κB subunit, p65, and their IL-1β/TNFα-induced reporter activity required consensus NF-κB motifs. Three such regions recruited RNA polymerase-2 and were flanked by the active chromatin mark, histone 3 lysine 27 acetylation, supporting enhancer involvement in IRF1 transcription. Finally, IRF1 expression, transcription rate, and enhancer activity induced by IL-1β, or TNFα, were relatively unaffected by glucocorticoid. IRF1-dependent gene expression may therefore show insensitivity to glucocorticoid and could contribute to glucocorticoid-resistance in diseases that include severe asthma.

Interferon (IFN)-I and IFN-II both induce IFN-stimulated gene (ISG) expression through Janus kinase (JAK)-dependent phosphorylation of signal transducer and activator of transcription (STAT) 1 and STAT2. STAT1 homodimers, known as γ-activated factor (GAF), activate transcription in response to all types of IFNs by direct binding to IFN-II activation site (γ-activated sequence)-containing genes. Association of interferon regulatory factor (IRF) 9 with STAT1-STAT2 heterodimers [known as interferon-stimulated gene factor 3 (ISGF3)] or with STAT2 homodimers (STAT2/IRF9) in response to IFN-I, redirects these complexes to a distinct group of target genes harboring the interferon-stimulated response element (ISRE). Similarly, IRF1 regulates expression of ISGs in response to IFN-I and IFN-II by directly binding the ISRE or IRF-responsive element. In addition, evidence is accumulating for an IFN-independent and -dependent role of unphosphorylated STAT1 and STAT2, with or without IRF9, and IRF1 in basal as well as long-term ISG expression. This review provides insight into the existence of an intracellular amplifier circuit regulating ISG expression and controlling long-term cellular responsiveness to IFN-I and IFN-II. The exact timely steps that take place during IFN-activated feedback regulation and the control of ISG transcription and long-term cellular responsiveness to IFN-I and IFN-II is currently not clear. Based on existing literature and our novel data, we predict the existence of a multifaceted intracellular amplifier circuit that depends on unphosphorylated and phosphorylated ISGF3 and GAF complexes and IRF1. In a combinatorial and timely fashion, these complexes mediate prolonged ISG expression and control cellular responsiveness to IFN-I and IFN-II. This proposed intracellular amplifier circuit also provides a molecular explanation for the existing overlap between IFN-I and IFN-II activated ISG expression.

The MHC class I-mediated antigen presentation pathway plays a critical role in antiviral immunity. Here we show that the MHC class I pathway is targeted by SARS-CoV-2. Analysis of the gene expression profile from COVID-19 patients as well as SARS-CoV-2 infected epithelial cell lines reveals that the induction of the MHC class I pathway is inhibited by SARS-CoV-2 infection. We show that NLRC5, an MHC class I transactivator, is suppressed both transcriptionally and functionally by the SARS-CoV-2 ORF6 protein, providing a mechanistic link. SARS-CoV-2 ORF6 hampers type II interferon-mediated STAT1 signaling, resulting in diminished upregulation of NLRC5 and IRF1 gene expression. Moreover, SARS-CoV-2 ORF6 inhibits NLRC5 function via blocking karyopherin complex-dependent nuclear import of NLRC5. Collectively, our study uncovers an immune evasion mechanism of SARS-CoV-2 that targets the function of key MHC class I transcriptional regulators, STAT1-IRF1-NLRC5. The presentation of viral antigens to T cells via the MHC molecules is a critical component of the host response to viral infection. Here the authors suggest SARS-CoV-2 possesses the immune evasion strategy against the MHC class I pathway by targeting key transcriptional regulators.

Keratinocytes are key barrier cells able to mount a robust interferon (IFN) antiviral response to defend against infection in the skin. Similar to humans, dogs spontaneously develop skin disorders associated with dysregulation of IFN immunity and can be used as a large animal model to investigate these diseases. One of the critical factors driving IFN regulation are interferon regulatory factors (IRFs). IRFs are crucial in upregulating the antiviral type I or type III IFNs, which then subsequently upregulate hundreds of antiviral effector proteins called interferon stimulated genes (ISGs). We sought to comparatively analyze how IRF1, 3 and 7 regulate type I and type III IFNs and ISGs using canine and human keratinocyte cultures. To this aim, we stimulated keratinocytes with dsDNA and dsRNA ligands and analyzed upregulation of IFNs and ISGs using real time quantitative PCR (RT-qPCR). Knockout of IRF3 and IRF1 and knockdown of IRF7 was performed on dog and human keratinocytes using CRISPR-Cas9 and shRNA methods, respectively. Knockdown or knockout was confirmed using RT-qPCR and/or western blots. We demonstrated that compared to canine keratinocytes, human keratinocytes express higher basal type I IFN-β and ISGs, induce higher levels of type III IFNs upon stimulation, and overall, express higher IFN and ISG copies. We further showed that IRF regulation of induced IFNs, particularly IRF1, can be species-specific and/or stimulus-specific, and putatively that non-IRF mechanisms likely mediate basal expression of type I IFNs in human keratinocytes and type III IFNs in canine keratinocytes. While both IRF3 and IRF7 are critical for type I and III IFN induction following activation of cytosolic dsDNA and dsRNA receptors, neither can account for differences in the induced type I versus III IFN expression between dog and human, suggesting non-IRF1, 3, or 7 mechanisms underly this regulation. These studies provide support for use of the dog as a model to discern mechanisms underlying high basal type I and ISGs in human keratinocytes, how high basal ISGs can modulate the antiviral response, and uncover non-IRF mediated mechanisms regulating the type I versus type III response in keratinocytes.