Induction Of IFN-stimulated Genes Research Articles

A PRMT5-ZNF326 axis mediates innate immune activation upon replication stress.

DNA replication stress (RS) is a widespread phenomenon in carcinogenesis, causing genomic instability and extensive chromatin alterations. DNA damage leads to activation of innate immune signaling, but little is known about transcriptional regulators mediating such signaling upon RS. Using a chemical screen, we identified protein arginine methyltransferase 5 (PRMT5) as a key mediator of RS-dependent induction of interferon-stimulated genes (ISGs). This response is also associated with reactivation of endogenous retroviruses (ERVs). Using quantitative mass spectrometry, we identify proteins with PRMT5-dependent symmetric dimethylarginine (SDMA) modification induced upon RS. Among these, we show that PRMT5 targets and modulates the activity of ZNF326, a zinc finger protein essential for ISG response. Our data demonstrate a role for PRMT5-mediated SDMA in the context of RS-induced transcriptional induction, affecting physiological homeostasis and cancer therapy.

Deciphering bat influenza H18N11 infection dynamics in male Jamaican fruit bats on a single-cell level.

Jamaican fruit bats (Artibeus jamaicensis) naturally harbor a wide range of viruses of human relevance. These infections are typically mild in bats, suggesting unique features of their immune system. To better understand the immune response to viral infections in bats, we infected male Jamaican fruit bats with the bat-derived influenza A virus (IAV) H18N11. Using comparative single-cell RNA sequencing, we generated single-cell atlases of the Jamaican fruit bat intestine and mesentery. Gene expression profiling showed that H18N11 infection resulted in a moderate induction of interferon-stimulated genes and transcriptional activation of immune cells. H18N11 infection was predominant in various leukocytes, including macrophages, B cells, and NK/T cells. Confirming these findings, human leukocytes, particularly macrophages, were also susceptible to H18N11, highlighting the zoonotic potential of this bat-derived IAV. Our study provides insight into a natural virus-host relationship and thus serves as a fundamental resource for future in-depth characterization of bat immunology.

Hepatocyte Intrinsic Innate Antiviral Immunity against Hepatitis Delta Virus Infection: The Voices of Bona Fide Human Hepatocytes.

The pathogenesis of viral infection is attributed to two folds: intrinsic cell death pathway activation due to the viral cytopathic effect, and immune-mediated extrinsic cellular injuries. The immune system, encompassing both innate and adaptive immunity, therefore acts as a double-edged sword in viral infection. Insufficient potency permits pathogens to establish lifelong persistent infection and its consequences, while excessive activation leads to organ damage beyond its mission to control viral pathogens. The innate immune response serves as the front line of defense against viral infection, which is triggered through the recognition of viral products, referred to as pathogen-associated molecular patterns (PAMPs), by host cell pattern recognition receptors (PRRs). The PRRs-PAMPs interaction results in the induction of interferon-stimulated genes (ISGs) in infected cells, as well as the secretion of interferons (IFNs), to establish a tissue-wide antiviral state in an autocrine and paracrine manner. Cumulative evidence suggests significant variability in the expression patterns of PRRs, the induction potency of ISGs and IFNs, and the IFN response across different cell types and species. Hence, in our understanding of viral hepatitis pathogenesis, insights gained through hepatoma cell lines or murine-based experimental systems are uncertain in precisely recapitulating the innate antiviral response of genuine human hepatocytes. Accordingly, this review article aims to extract and summarize evidence made possible with bona fide human hepatocytes-based study tools, along with their clinical relevance and implications, as well as to identify the remaining gaps in knowledge for future investigations.

Retraction: Induction of Interferon-Stimulated Genes by IRF3 Promotes Replication of Toxoplasma gondii.

Retraction: Induction of Interferon-Stimulated Genes by IRF3 Promotes Replication of Toxoplasma gondii.

Differential innate immune responses to fowl adenovirus serotype 4 infection in Leghorn male hepatocellular and chicken embryo fibroblast cells

Fowl adenovirus serotype 4 (FAdV-4) infections result in substantial economic losses in the poultry industry. Recent findings have revealed that FAdV-4 significantly suppresses the host immune response upon infection; however, the specific viral and host factors contributing to this immunomodulatory activity remain poorly characterized. Moreover, diverse cell types exhibit differential immune responses to FAdV-4 infection. To elucidate cell-specific host responses, we performed transcriptomic analysis of FAdV-4 infected leghorn male hepatocellular (LMH) and chicken embryo fibroblast (CEF) cells. Although FAdV-4 replicated more efficiently in LMH cells, it provoked limited interferon-stimulated gene induction. In contrast, FAdV-4 infection triggered robust antiviral responses in CEF cells, including upregulation of cytosolic DNA sensing and interferon-stimulated genes. Knockdown of key cytosolic DNA sensing molecules enhanced FAdV-4 replication in LMH cells while reducing interferon-stimulated gene expression. Our findings reveal cell-specific virus-host interactions that provide insight into FAdV-4 pathogenesis while identifying factors that mediate antiviral immunity against FAdV-4.

Gasdermin B, an asthma-susceptibility gene, promotes MAVS-TBK1 signalling and airway inflammation.

Respiratory virus-induced inflammation is the leading cause of asthma exacerbation, frequently accompanied by induction of interferon-stimulated genes (ISGs). How asthma-susceptibility genes modulate cellular response upon viral infection by fine-tuning ISG induction and subsequent airway inflammation in genetically susceptible asthma patients remains largely unknown. To decipher the functions of gasdermin B (encoded by GSDMB) in respiratory virus-induced lung inflammation. In two independent cohorts, we analysed expression correlation between GSDMB and ISG s. In human bronchial epithelial cell line or primary bronchial epithelial cells, we generated GSDMB-overexpressing and GSDMB-deficient cells. A series of quantitative PCR, ELISA and co-immunoprecipitation assays were performed to determine the function and mechanism of GSDMB for ISG induction. We also generated a novel transgenic mouse line with inducible expression of human unique GSDMB gene in airway epithelial cells and infected the mice with respiratory syncytial virus to determine the role of GSDMB in respiratory syncytial virus-induced lung inflammation in vivo. GSDMB is one of the most significant asthma-susceptibility genes at 17q21 and acts as a novel RNA sensor, promoting mitochondrial antiviral-signalling protein (MAVS)-TANK binding kinase 1 (TBK1) signalling and subsequent inflammation. In airway epithelium, GSDMB is induced by respiratory viral infections. Expression of GSDMB and ISGs significantly correlated in respiratory epithelium from two independent asthma cohorts. Notably, inducible expression of human GSDMB in mouse airway epithelium led to enhanced ISGs induction and increased airway inflammation with mucus hypersecretion upon respiratory syncytial virus infection. GSDMB promotes ISGs expression and airway inflammation upon respiratory virus infection, thereby conferring asthma risk in risk allele carriers.

Induction of antiviral interferon-stimulated genes by neuronal STING promotes the resolution of pain in mice.

Inflammation and pain are intertwined responses to injury, infection, or chronic diseases. While acute inflammation is essential in determining pain resolution and opioid analgesia, maladaptive processes occurring during resolution can lead to the transition to chronic pain. Here we found that inflammation activates the cytosolic DNA-sensing protein stimulator of IFN genes (STING) in dorsal root ganglion nociceptors. Neuronal activation of STING promotes signaling through TANK-binding kinase 1 (TBK1) and triggers an IFN-β response that mediates pain resolution. Notably, we found that mice expressing a nociceptor-specific gain-of-function mutation in STING exhibited an IFN gene signature that reduced nociceptor excitability and inflammatory hyperalgesia through a KChIP1-Kv4.3 regulation. Our findings reveal a role of IFN-regulated genes and KChIP1 downstream of STING in the resolution of inflammatory pain.

Interferon-β deficiency alters brain response to chronic HIV-1 envelope protein exposure in a transgenic model of NeuroHIV

Human immunodeficiency virus-1 (HIV-1) infects the central nervous system (CNS) and causes HIV-associated neurocognitive disorders (HAND) in about half of the population living with the virus despite combination anti-retroviral therapy (cART). HIV-1 activates the innate immune system, including the production of type 1 interferons (IFNs) α and β. Transgenic mice expressing HIV-1 envelope glycoprotein gp120 (HIVgp120tg) in the CNS develop memory impairment and share key neuropathological features and differential CNS gene expression with HIV patients, including the induction of IFN-stimulated genes (ISG). Here we show that knocking out IFNβ (IFNβKO) in HIVgp120tg and non-tg control mice impairs recognition and spatial memory, but does not affect anxiety-like behavior, locomotion, or vision. The neuropathology of HIVgp120tg mice is only moderately affected by the KO of IFNβ but in a sex-dependent fashion. Notably, in cerebral cortex of IFNβKO animals presynaptic terminals are reduced in males while neuronal dendrites are reduced in females. The IFNβKO results in the hippocampal CA1 region of both male and female HIVgp120tg mice in an ameliorated loss of neuronal presynaptic terminals but no protection of neuronal dendrites. Only female IFNβ-deficient HIVgp120tg mice display diminished microglial activation in cortex and hippocampus and increased astrocytosis in hippocampus compared to their IFNβ-expressing counterparts. RNA expression for some immune genes and ISGs is also affected in a sex-dependent way. The IFNβKO abrogates or diminishes the induction of MX1, DDX58, IRF7 and IRF9 in HIVgp120tg brains of both sexes. Expression analysis of neurotransmission related genes reveals an influence of IFNβ on multiple components with more pronounced changes in IFNβKO females. In contrast, the effects of IFNβKO on MAPK activities are independent of sex with pronounced reduction of active ERK1/2 but also of active p38 in the HIVgp120tg brain. In summary, our findings show that the absence of IFNβ impairs memory dependent behavior and modulates neuropathology in HIVgp120tg brains, indicating that its absence may facilitate development of HAND. Moreover, our data suggests that endogenous IFNβ plays a vital role in maintaining neuronal homeostasis and memory function.

Liver-restricted Type I IFN Signature Precedes Liver Damage in Chronic Hepatitis B Patients Stopping Antiviral Therapy.

Immune-mediated liver damage is the driver of disease progression in patients with chronic hepatitis B virus (HBV) infection. Liver damage is an Ag-independent process caused by bystander activation of CD8 T cells and NK cells. How bystander lymphocyte activation is initiated in chronic hepatitis B patients remains unclear. Periods of liver damage, called hepatic flares, occur unpredictably, making early events difficult to capture. To address this obstacle, we longitudinally sampled the liver of chronic hepatitis B patients stopping antiviral therapy and analyzed immune composition and activation using flow cytometry and single-cell RNA sequencing. At 4 wk after stopping therapy, HBV replication rebounded but no liver damage was detectable. There were no changes in cell frequencies at viral rebound. Single-cell RNA sequencing revealed upregulation of IFN-stimulated genes (ISGs) and proinflammatory cytokine migration inhibitory factor (MIF) at viral rebound in patients that go on to develop hepatic flares 6-18 wk after stopping therapy. The type I IFN signature was only detectable within the liver, and neither IFN-α/β or ISG induction could be detected in the peripheral blood. In vitro experiments confirmed the type I IFN-dependent ISG profile whereas MIF was induced primarily by IL-12. MIF exposure further amplified inflammatory cytokine production by myeloid cells. Our data show that innate immune activation is detectable in the liver before clinically significant liver damage is evident. The combination of type I IFN and enhanced cytokine production upon MIF exposure represent the earliest immunological triggers of lymphocyte bystander activation observed in hepatic flares associated with chronic HBV infection.

Candida albicans extracellular vesicles trigger type I IFN signalling via cGAS and STING.

The host type I interferon (IFN) pathway is a major signature of inflammation induced by the human fungal pathogen, Candida albicans. However, the molecular mechanism for activating this pathway in the host defence against C. albicans remains unknown. Here we reveal that mice lacking cyclic GMP-AMP synthase (cGAS)-stimulator of IFN genes (STING) pathway components had improved survival following an intravenous challenge by C. albicans. Biofilm-associated C. albicans DNA packaged in extracellular vesicles triggers the cGAS-STING pathway as determined by induction of interferon-stimulated genes, IFNβ production, and phosphorylation of IFN regulatory factor 3 and TANK-binding kinase 1. Extracellular vesicle-induced activation of type I IFNs was independent of the Dectin-1/Card9 pathway and did not require toll-like receptor 9. Single nucleotide polymorphisms in cGAS and STING potently altered inflammatory cytokine production in human monocytes challenged by C. albicans. These studies provide insights into the early innate immune response induced by a clinically significant fungal pathogen.

The Asthma Risk Gene, GSDMB, Promotes Mitochondrial DNA-induced ISGs Expression.

Released mitochondrial DNA (mtDNA) in cells activates cGAS-STING pathway, which induces expression of interferon-stimulated genes (ISGs) and thereby promotes inflammation, as frequently seen in asthmatic airways. However, whether the genetic determinant, Gasdermin B (GSDMB), the most replicated asthma risk gene, regulates this pathway remains unknown. We set out to determine whether and how GSDMB regulates mtDNA-activated cGAS-STING pathway and subsequent ISGs induction in human airway epithelial cells. Using qPCR, ELISA, native polyacrylamide gel electrophoresis, co-immunoprecipitation and immunofluorescence assays, we evaluated the regulation of GSDMB on cGAS-STING pathway in both BEAS-2B cells and primary normal human bronchial epithelial cells (nHBEs). mtDNA was extracted in plasma samples from human asthmatics and the correlation between mtDNA levels and eosinophil counts was analyzed. GSDMB is significantly associated with RANTES expression in asthmatic nasal epithelial brushing samples from the Genes-environments and Admixture in Latino Americans (GALA) II study. Over-expression of GSDMB promotes DNA-induced IFN and ISGs expression in bronchial epithelial BEAS-2B cells and nHBEs. Conversely, knockout of GSDMB led to weakened induction of interferon (IFNs) and ISGs in BEAS-2B cells. Mechanistically, GSDMB interacts with the C-terminus of STING, promoting the translocation of STING to Golgi, leading to the phosphorylation of IRF3 and induction of IFNs and ISGs. mtDNA copy number in serum from asthmatics was significantly correlated with blood eosinophil counts especially in male subjects. GSDMB promotes the activation of mtDNA and poly (dA:dT)-induced activation of cGAS-STING pathway in airway epithelial cells, leading to enhanced induction of ISGs.

Sox10-Deficient Drug-Resistant Melanoma Cells Are Refractory to Oncolytic RNA Viruses.

Targeted therapy resistance frequently develops in melanoma due to intratumor heterogeneity and epigenetic reprogramming. This also typically induces cross-resistance to immunotherapies. Whether this includes additional modes of therapy has not been fully assessed. We show that co-treatments of MAPKi with VSV-based oncolytics do not function in a synergistic fashion; rather, the MAPKis block infection. Melanoma resistance to vemurafenib further perturbs the cells' ability to be infected by oncolytic viruses. Resistance to vemurafenib can be induced by the loss of SOX10, a common proliferative marker in melanoma. The loss of SOX10 promotes a cross-resistant state by further inhibiting viral infection and replication. Analysis of RNA-seq datasets revealed an upregulation of interferon-stimulated genes (ISGs) in SOX10 knockout populations and targeted therapy-resistant cells. Interestingly, the induction of ISGs appears to be independent of type I IFN production. Overall, our data suggest that the pathway mediating oncolytic resistance is due to the loss of SOX10 during acquired drug resistance in melanoma.

Genome-wide and targeted CRISPR screens identify RNF213 as a mediator of interferon gamma-dependent pathogen restriction in human cells.

To define cellular immunity to the intracellular pathogen Toxoplasma gondii, we performed a genome-wide CRISPR loss-of-function screen to identify genes important for (interferon gamma) IFN-γ-dependent growth restriction. We revealed a role for the tumor suppressor NF2/Merlin for maximum induction of Interferon Stimulated Genes (ISG), which are positively regulated by the transcription factor IRF-1. We then performed an ISG-targeted CRISPR screen that identified the host E3 ubiquitin ligase RNF213 as necessary for IFN-γ-mediated control of T. gondii in multiple human cell types. RNF213 was also important for control of bacterial (Mycobacterium tuberculosis) and viral (Vesicular Stomatitis Virus) pathogens in human cells. RNF213-mediated ubiquitination of the parasitophorous vacuole membrane (PVM) led to growth restriction of T. gondii in response to IFN-γ. Moreover, overexpression of RNF213 in naive cells also impaired growth of T. gondii. Surprisingly, growth inhibition did not require the autophagy protein ATG5, indicating that RNF213 initiates restriction independent of a previously described noncanonical autophagy pathway. Mutational analysis revealed that the ATPase domain of RNF213 was required for its recruitment to the PVM, while loss of a critical histidine in the RZ finger domain resulted in partial reduction of recruitment to the PVM and complete loss of ubiquitination. Both RNF213 mutants lost the ability to restrict growth of T. gondii, indicating that both recruitment and ubiquitination are required. Collectively, our findings establish RNF213 as a critical component of cell-autonomous immunity that is both necessary and sufficient for control of intracellular pathogens in human cells.

Recombinant porcine interferon-gamma expressed in CHO cells and its antiviral activity

The aim of this study was to produce recombinant porcine interferon gamma (rPoIFN-γ) by Chinese hamster ovarian (CHO) cells expression system and to analyze its antiviral activity. Firstly, we constructed the recombinant eukaryotic expression plasmid pcDNA3.1-PoIFN-γ and transfected into suspension cultured CHO cells for secretory expression of rPoIFN-γ. The rPoIFN-γ was purified by affinity chromatography and identified with SDS-PAGE and Western blotting. Subsequently, the cytotoxicity of rPoIFN-γ was analyzed by CCK-8 test, and the antiviral activity of rPoIFN-γ was evaluated using standard procedures in VSV/PK-15 (virus/cell) test system. Finally the anti-Seneca virus A (SVA) of rPoIFN-γ activity and the induction of interferon-stimulated genes (ISGs) and cytokines were also analyzed. The results showed that rPoIFN-γ could successfully expressed in the supernatant of CHO cells. CCK-8 assays indicated that rPoIFN-γ did not show cytotoxicity on IBRS-2 cells. The biological activity of rPoIFN-γ was 5.59×107 U/mg in VSV/PK-15 system. Moreover, rPoIFN-γ could induced the expression of ISGs and cytokines, and significantly inhibited the replication of SVA. In conclusion, the high activity of rPoIFN-γ was successfully prepared by CHO cells expression system, which showed strong antiviral activity on SVA. This study may facilitate the investigation of rPoIFN-γ function and the development of novel genetically engineered antiviral drugs.

Targeting EIF4A triggers an interferon response to synergize with chemotherapy and suppress triple-negative breast cancer.

Protein synthesis is frequently dysregulated in cancer and selective inhibition of mRNA translation represents an attractive cancer therapy. Here, we show that therapeutically targeting the RNA helicase eIF4A by Zotatifin, the first-in-class eIF4A inhibitor, exerts pleiotropic effects on both tumor cells and the tumor immune microenvironment in a diverse cohort of syngeneic triple-negative breast cancer (TNBC) mouse models. Zotatifin not only suppresses tumor cell proliferation but also directly repolarizes macrophages towards an M1-like phenotype and inhibits neutrophil infiltration, which sensitizes tumors to immune checkpoint blockade. Mechanistic studies revealed that Zotatifin reprograms the tumor translational landscape, inhibits the translation of Sox4 and Fgfr1, and induces an interferon response uniformly across models. The induction of an interferon response is partially due to the inhibition of Sox4 translation by Zotatifin. A similar induction of interferon-stimulated genes was observed in breast cancer patient biopsies following Zotatifin treatment. Surprisingly, Zotatifin significantly synergizes with carboplatin to trigger DNA damage and an even heightened interferon response resulting in T cell-dependent tumor suppression. These studies identified a vulnerability of eIF4A in TNBC, potential pharmacodynamic biomarkers for Zotatifin, and provide a rationale for new combination regimens comprising Zotatifin and chemotherapy or immunotherapy as treatments for TNBC.

Young infants display heterogeneous serological responses and extensive but reversible transcriptional changes following initial immunizations

Infants necessitate vaccinations to prevent life-threatening infections. Our understanding of the infant immune responses to routine vaccines remains limited. We analyzed two cohorts of 2-month-old infants before vaccination, one week, and one-month post-vaccination. We report remarkable heterogeneity but limited antibody responses to the different antigens. Whole-blood transcriptome analysis in an initial cohort showed marked overexpression of interferon-stimulated genes (ISGs) and to a lesser extent of inflammation-genes at day 7, which normalized one month post-vaccination. Single-cell RNA sequencing in peripheral blood mononuclear cells from a second cohort identified at baseline a predominantly naive immune landscape including ISGhi cells. On day 7, increased expression of interferon-, inflammation-, and cytotoxicity-related genes were observed in most immune cells, that reverted one month post-vaccination, when a CD8+ ISGhi and cytotoxic cluster and B cells expanded. Antibody responses were associated with baseline frequencies of plasma cells, B-cells, and monocytes, and induction of ISGs at day 7.

Insights on interferon-independent induction of interferon-stimulated genes shaping the lung's response in early SARS-CoV-2 infection

While mRNA vaccine efficacy against the 2019 coronavirus disease (COVID-19) outbreak remains high, research on antiviral innate immune responses in the early stages of infection is essential to develop strategies to prevent the dissemination of SARS-CoV-2. In this study, we investigated the induction of both interferon (IFN)-stimulated genes (ISGs) and IFN-independently upregulated ISGs following SARS-CoV-2 infection in Syrian golden hamsters. The viral titers were highest at 3 days post-infection (dpi). Over time, the viral titer gradually decreased while ISGs such as Mx1, Ifit2, Ifit3, Ifi44, and Rsad2 were markedly induced in the lung. The transcription of ISGs significantly increased from 2 dpi, and SARS-CoV-2-induced ISGs were maintained in the hamster lung until 7 dpi. The transcription of Ifnb and Ifng was minimally elevated, while Ifnl2/3 was significantly induced in the lung at 5 days after SARS-CoV-2 infection. RNA sequencing results also showed that at 3 dpi, SARS-CoV-2 initiated the activation of ISGs, with lesser increases of Ifnl2 and Ifnl3 transcription. In addition, Ddx58 and cGAS, which encode factors for virus sensing, Stat1, Stat2, and IFN regulatory factor 7 and 9 mRNA levels were also induced at the initial stage of infection. Our data demonstrate that ISGs might be upregulated in the lung in response to SARS-CoV-2 during the early stages of infection, and the rapid induction of ISGs was not associated with the activation of IFNs. Elucidation of IFN-independent induction of ISGs could further our understanding of alternative defense mechanisms employed by the lungs against SARS-CoV-2 and provide more effective antiviral strategies for patients with severe COVID-19.

Sting Activation Improves T-Cell Engaging Immunotherapy of Acute Myeloid Leukemia

Background: Despite emerging therapeutic options for Acute myeloid leukemia (AML) patients, for the majority of relapsed/refractory patients the outcome remains poor. Allogeneic stem cell transplantation has proven the power of T cells in eradicating residual leukemic cells, but alternative strategies based on T-cell recruiting bispecifics have so far failed to induce sustained responses. Resistance is mediated by the immunosuppressive tumor-microenvironment and secretion of immune dampening metabolites by AML cells. We hypothesized that combining a CD33-directed T cell engager (CD33 BiTE ® molecule or AMG 330) with a cGAS-STING agonist has the potential to augment anti-leukemic activity. Methods: In vitro co-culture assays of human T cells and AML cell lines for up to 72 h were performed to study the effect of AMG 330 in combination with the physiological STING-agonist 2‘-3‘ cGAMP (cGAMP). The cytotoxicity against the AML cell lines HL-60, MOLM-13 and OCI-AML-3 was assessed by flow cytometry. T-cell proliferation, T-cell degranulation (CD107a), as well as Granzyme B and TRAIL expression were characterized by flow cytometry. The secretion of effector cytokines (IFNy, TNF, IL-2, IFNa2a, CXCL10) was analyzed via CBA and ELISA assays. To better understand how AMG 330 and cGAMP work together, immunoblotting, transcriptomic analysis (bulk RNA-sequencing) and comprehensive CRISPRCas9 knockout-studies were carried out. Results: We observed a significant increase in AMG 330-mediated cytotoxicity of AML target cells upon co-application of cGAMP (Fig1A). The latter observation was enhanced upon prolongation of the culture and was also notable at low effector-to-target ratios. Monotherapy or addition of cGAMP by itself exerted only minor cytotoxicity against AML cells. The increase in AMG 330 mediated cytotoxicity through cGAMP was accompanied by an increase in T-cell proliferation, degranulation, Granzyme B secretion and TRAIL expression. Moreover, we observed a strong increase in secretion of proinflammatory cytokines (IFNy, IL-2 and IFNa2a). Bulk RNA-sequencing of AMG 330 and cGAMP stimulated co-cultures revealed type-I-IFN signature in the target (HL-60) and the effector cell (T cells) cell population. In addition, distinct phenotypes were found in each cell population: T cells demonstrated a marked increase in TNFa, IFNy and GZMB expression through the co-treatment, whereas HL-60 cells displayed an IFNy signaling signature (Fig1B). Importantly, the responsiveness of AML target cells to STING activation in terms of interferon stimulated gene (ISG)-induction was increased by the co-treatment, evidenced by further upregulation of STAT1, IRF7, WARS, CXCL11, IFI16, RSAD2 and CXCL10. An increase in type-I-IFN production and subsequent ISG-induction could also be confirmed on the protein level by measuring IFNα2a and CXCL-10. Knockout-studies revealed that the cGAMP enhanced AMG 330-mediated cytotoxicity was fully dependent on target cell intrinsic STING and IRF3 signaling, but also dependent on functional IFNy and TNFa signaling. Notably, the increased IFNy production by T cells upon cGAMP addition was dependent on functional target cell STING and IFNy signaling. These observations imply a crosstalk between effector and target cells and an intricate interdependence between target cell intrinsic IFNγ- and STING-signaling. Of note, the deletion of the IFNy-receptor in AML target cells also reduced AMG 330-dependent cytotoxicity under steady state conditions. Finally, we demonstrated that the combination of IFNy, TNFa and cGAMP sufficed to induce an apoptotic cell death of AML cells, similar to the cGAMP containing T-cell-HL-60 cocultures. Conclusion: We propose a novel mechanism by which AMG 330-activated T cells prime and sensitize AML target cells in a forward feedback loop towards STING activation, leading to increased type-I-IFN production and induction of ISGs. The beneficial effect of physiological cGAMP in enhancing AMG 330-mediated cytotoxicity was accompanied by the pronounced expression of effector cytokines and an overall cytotoxic T-cell phenotype. We established a key role for interferon gamma in AMG 330-mediated cytotoxicity of AML cells and in rendering AML cells responsive to STING agonism. We are currently conducting in vivoexperiments to further validate our findings.

Interferon regulatory factor 7 modulates virus clearance and immune responses to alphavirus encephalomyelitis.

Viral encephalomyelitis outcome is dependent on host responses to neuronal infection. Interferon (IFN) is an important component of the innate response, and IFN regulatory factor (IRF) 7 is an inducible transcription factor for the synthesis of IFN-α. IRF7-deficient mice develop fatal paralysis after CNS infection with Sindbis virus, while wild-type mice recover. Irf7 -/- mice produce low levels of IFN-α but high levels of IFN-β with induction of IFN-stimulated genes, so the reason for this difference is not understood. The current study shows that Irf7 -/- mice developed inflammation earlier but failed to clear virus from motor neuron-rich regions of the brainstem and spinal cord. Levels of IFN-γ and virus-specific antibody were comparable, indicating that IRF7 deficiency does not impair expression of these known viral clearance factors. Therefore, IRF7 is either necessary for the neuronal response to currently identified mediators of clearance or enables the production of additional antiviral factor(s) needed for clearance.

Interferon-λ Activates a Differential Response in Peripheral Neurons That Is Effective against Alpha Herpesvirus Infections.

Alpha herpesviruses (α-HV) infect host mucosal epithelial cells prior to establishing a life-long latent infection in the peripheral nervous system. The initial spread of viral particles from mucosa to the nervous system and the role of intrinsic immune responses at this barrier is not well understood. Using primary neurons cultured in compartmentalized chambers, prior studies performed on Pseudorabies virus (PRV) have demonstrated that type I and type II interferons (IFNs) induce a local antiviral response in axons via distinct mechanisms leading to a reduction in viral particle transport to the neuronal nucleus. A new class of interferons known as type III IFNs has been shown to play an immediate role against viral infection in mucosal epithelial cells. However, the antiviral effects of type III IFNs within neurons during α-HV infection are largely unknown. In this study, we focused on elucidating the antiviral activity of type III IFN against PRV neuronal infection, and we compared the interferon-stimulated gene (ISGs) induction pattern in neurons to non-neuronal cells. We found that IFN pre-exposure of both primary neurons and fibroblast cells significantly reduces PRV virus yield, albeit by differential STAT activation and ISG induction patterns. Notably, we observed that type III IFNs trigger the expression of a subset of ISGs mainly through STAT1 activation to induce an antiviral state in primary peripheral neurons.