Transcription Of IFN-stimulated Genes Research Articles

Long non-coding RNA SUN2-AS1 acts as a negative regulator of ISGs transcription to promote flavivirus infection

Recent studies highlight the critical involvement of long non-coding RNAs (lncRNAs) in modulating viral replication and immune responses, yet their specific roles in flavivirus infections remain underexplored. Our study has identified lncRNA SUN2-AS1, which is significantly upregulated in response to flavivirus infection in A549, Huh7 cells, and monocyte-differentiated macrophages (MDMs). SUN2-AS1 interacts with the transcription factors NF-κB and STAT1, andits expression is induced by ZIKV RNA via the type I interferon (IFN) pathway. Notably, SUN2-AS1 enhances the infection of flaviviruses, including ZIKV, DENV2, and JEV, while showing no effect on VSV or HSV-1 infections. Mechanistically, SUN2-AS1 exerts a proviral effect by inhibiting the transcription of interferon-stimulated genes (ISGs). These findings uncover a novel mechanism by which lncRNAs facilitate flavivirus propagation and highlight SUN2-AS1 as a potential target for antiviral therapeutic strategies.

Transcriptomic analysis reveals bovine herpesvirus 1 infection regulates innate immune response resulted in restricted viral replication in neuronal cells

BackgroundBovine herpesvirus 1 (BoHV-1) is a major pathogen that affects the global bovine population, primarily inducing respiratory and reproductive disorders. Its ability to establish latent infections in neuronal cells and to reactivate under certain conditions poses a continual threat to uninfected hosts. In this study, we aimed to analyze the replication characteristics of BoHV-1 in neuronal cells, as well as the effects of viral replication on host cell immunity and physiology. MethodsUsing the Neuro-2a neuronal-origin cell line as a model, we explored the dynamics of BoHV-1 replication and analyzed differential gene expression profiles post-BoHV-1 infection using high-throughput RNA sequencing. ResultsBoHV-1 demonstrated restricted replication in Neuro-2a cells. BoHV-1 induced apoptotic pathways and enhanced the transcription of interferon-stimulated genes and interferon regulatory factors while suppressing the complement cascade in Neuro-2a cells. ConclusionsDifferent from BoHV-1 infection in other non-highly differentiated somatic cells result in viral dominance, BoHV-1 regulated the innate immune response in neuronal cells formed a “virus-nerve cell” relative equilibrium state, which may account for the restricted replication of BoHV-1 in neuronal cells, leading to a latent infection. These findings provide a foundation for further research into the mechanism underlying BoHV-1-induced latent infection in nerve cells.

Increased serum interferon activity in sarcoidosis compared to that in tuberculosis: Implication for diagnosis?

ObjectivesIn this study, we measured serum interferon (IFN) levels and activity in patients with sarcoidosis and tuberculosis (TB) with and without uveitis. We aimed to understand the role of IFN in the pathophysiology of both conditions and explore its potential as a discriminating marker for these clinically similar diseases. MethodsSera from an Indonesian TB and a Dutch sarcoidosis cohort were used in the analysis. IFNα2 and IFNγ concentrations were measured using Simoa® and Luminex assays, respectively. Serum IFN activity was assessed by incubating THP-1 cells with patient serum and measuring IFN-stimulated gene transcription using qPCR. Anti-IFNα2 and IFNγ autoantibodies were detected via Luminex assay and tested for neutralizing capacity using a flow cytometry-based signal transducer and activator of transcription (STAT) 1 phosphorylation inhibition assay. ResultsIFNα2 was detected in 74 % and 64 % of patients with sarcoidosis and pulmonary TB, respectively, while IFNγ was found in 78 % and 23 % of patients with sarcoidosis and TB, respectively. For uveitis cases specifically, IFNα2 was detected in 85 % of sarcoid uveitis (SU) and 33 % of tubercular uveitis (TBU) cases. Similarly, IFNγ was detected in 69 % of SU and 17 % of TBU cases. IFNγ serum concentrations were higher in sarcoidosis than that in TB patients (p < 0.0001). Focusing on patients with uveitis, SU showed increased IFNα2 (p = 0.004) and IFNγ (p < 0.002) serum concentrations compared to that in TBU. Notably, TBU displayed significantly reduced IFNα2 concentrations compared to that in healthy controls (p = 0.006). These results align with the increased interferon stimulated gene (ISG) transcriptional upregulation observed in THP-1 cells stimulated with serum from patients with sarcoidosis. Elevated levels of non-neutralizing anti-IFN autoantibodies were observed in patients with TB; however, these levels were similar to those observed in geographically matched healthy Indonesian controls. ConclusionOur results suggest decreased serum levels and activity of type I and II IFN in TB compared to those in sarcoidosis. This is indicative of distinct pathophysiological processes in these highly clinically similar diseases. We propose that the assessment of serum IFN levels and IFN activity has the potential to distinguish between sarcoidosis/SU and TB/TBU.

Innate immune signatures in the nasopharynx after SARS-CoV-2 infection and links with the clinical outcome of COVID-19 in Omicron-dominant period

While severe acute respiratory syndrome coronavirus-2 (SARS-CoV-2) is characterized by impaired induction of interferons (IFNs) and IFN-stimulated genes (ISGs), the IFNs and ISGs in upper airway is essential to restrict the spread of respiratory virus. Here, we identified the prominent IFN and ISG upregulation in the nasopharynx (NP) of mild and even severe coronavirus disease 2019 (COVID-19) patients (CoV2+) in Omicron era and to compare their clinical outcome depending on the level of IFNs and ISGs. Whereas the induction of IFNB was minimal, transcription of IFNA, IFNG, and IFNLs was significantly increased in the NP of CoV2 + patients. IFNs and ISGs may be more upregulated in the NP of CoV2 + patients at early phases of infection according to viral RNA levels and this is observed even in severe cases. IFN-related innate immune response might be characteristic in macrophages and monocytes at the NP and the CoV2 + patients with higher transcription of IFNs and ISGs in the NP showed a correlation with good prognosis of COVID-19. This study presents that IFNs and ISGs may be upregulated in the NP, even in severe CoV2 + patients depending on viral replication during Omicron-dominant period and the unique IFN-responsiveness in the NP links with COVID-19 clinical outcomes.

Research progress on interferon and cellular senescence.

Since the 12 major signs of aging were revealed in 2023, people's interpretation of aging will go further, which is of great significance for understanding the occurrence, development, and intervention in the aging process. As one of the 12 major signs of aging, cellular senescence refers to the process in which the proliferation and differentiation ability of cells decrease under stress stimulation or over time, often manifested as changes in cell morphology, cell cycle arrest, and decreased metabolic function. Interferon (IFN), as a secreted ligand for specific cell surface receptors, can trigger the transcription of interferon-stimulated genes (ISGs) and play an important role in cellular senescence. In addition, IFN serves as an important component of SASP, and the activation of the IFN signaling pathway has been shown to contribute to cell apoptosis and senescence. It is expected to delay cellular senescence by linking IFN with cellular senescence and studying the effects of IFN on cellular senescence and its mechanism. This article provides a review of the research on the relationship between IFN and cellular senescence by consulting relevant literature.

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.

Phase II Clinical Trial of R-CHOP Combined with Lenalidomide in the First-Line Treatment for Patients with High Risk Diffuse Large B Cell Lymphoma

Introduction: Diffuse large B cell lymphoma (DLBCL) is the most common type of non-Hodgkin's lymphoma (NHL). Currently, R-CHOP (Rituximab-Cyclophosphamide, Epirubicin, Vincristine and Prednisone) is world-widely used in the first-line treatment for DLBCL, producing a long-term survival rate of approximately 60%. The unmet need is focused on high-risk DLBCL patients, having high International Prognostic Index (IPI) scores, non-germinal center-like B cell (non-GCB) origin, double expression of MYC and BCL2 protein, or TP53 aberrations. Lenalidomide is an active agent in the activated B cell-like (ABC) subtype of DLBCL by increasing interferon-stimulated gene transcription and activation of immunomodulatory mechanisms. At present, it has been approved for the treatment of multiple myeloma with good efficacy and safety. This is a prospective single arm, multi-center, phase II clinical trial and the goal of our trial is to assess the efficacy and safety of R-CHOP combined with lenalidomide in the first-line treatment for patients with high risk diffuse large B cell lymphoma. Methods: We did an investigator-initiated, open-label, phase 2 trial at the Affiliated Cancer Hospital of Zhengzhou University in China. Patients were eligible if they were aged over 18 years, had histologically confirmed DLBCL. The key inclusion criteria included untreated and medium to high /high risk DLBCL (International Prognostic Index (IPI) score 3-5, aaIPI score 2-3 or NCCN-IPI score≥ 4/ Immunohistochemical staining of double expression (BCL2 ≥ 50% and C-MYC ≥ 40%) or P53 protein mutation positive ≥ 50%). Participants in RL-CHOP cohort were given Lenalidomide orally 25 mg per day on days 1 through 10 of each cycle and delivered concomitantly with standard dose R-CHOP-21 chemotherapy. We also set another group treated with conventional R-CHOP-21 used as a matched contemporary cohort. The primary clinical endpoint was 2-year progression-free survival (PFS). The secondary efficacy endpoints were objective response rate (ORR), 2-year overall survival (OS) and safety. This trial is registered with ClinicalTrials.gov, number NCT04214626. Results: Between December 2019 and January 2023, 63 patients with untreated DLBCL enrolled and were evaluable in the investigator-initiated phase II study. The median age was 59 years (range: 25-76 years); 49.2% of patients were older than age 60 years. 46 of 63 patients (73.02%) had a ECOG score of 0-1; 43 of 63 patients (68.25%) had stage III-IV disease, and 33 of 63 patients (52.38%) had ≥2 extranodal localizations. The IPI score was high-intermediate and high in 73.02% (46/63) of patients. 51 of 63 patients (80.95%) were non-GCB subtype; 25 of 63 patients (39.68%) were double expressor. The TP53 protein was positive in 34.55% (19/55) of patients. In RL-CHOP cohort, the complete response rate was 80.95%, with overall response rate achieving 100%. The median follow-up time was 18.25 (3.73-42) months, median PFS and OS were not reached, and 2-year PFS and OS was 71.1% (+-6.7) and 87.4% (+-4.5), respectively. Comparing with historical matched contemporary cohort, the 2-year PFS and OS rates were improved for medium to high /high risk DLBCL. The most common adverse events of treatment were hematologic toxicities. Grade 3/4 AEs occurred in ≥20% patients were neutropenia, leukopenia, anemia, thrombocytopenia and decreased serum potassium, without bleeding complications. No grade 4 non-hematological adverse event was reported. Conclusion: RL-CHOP is effective and safe in patients with newly diagnosed DLBCL with high risk. Relevance of molecular biomarkers on RL-CHOP response warrants further investigation in DLBCL.

Enterovirus D68 infection upregulates SOCS3 expression to inhibit JAK-STAT3 signaling and antagonize the innate interferon response of the host

Enterovirus D68 (EV-D68) can cause respiratory diseases and acute flaccid paralysis, posing a great threat to public health. Interferons are cytokines secreted by host cells that have broad-spectrum antiviral effects, inducing the expression of hundreds of interferon-stimulated genes (ISGs). EV-D68 activates ISG expression early in infection, but at a later stage, the virus suppresses ISG expression, a strategy evolved by EV-D68 to antagonize interferons. Here, we explore a host protein, suppressor of cytokine signaling 3 (SOCS3), is upregulated during EV-D68 infection and antagonizes the antiviral effects of type I interferon. We subsequently demonstrate that the structural protein of EV-D68 upregulated the expression of RFX7, a transcriptional regulator of SOCS3, leading to the upregulation of SOCS3 expression. Further exploration revealed that SOCS3 plays its role by inhibiting the phosphorylation of signal transducer and activator of transcription 3 (STAT3). The expression of SOCS3 inhibited the expression of ISG, thereby inhibiting the antiviral effect of type I interferon and promoting EV-D68 transcription, protein production, and viral titer. Notably, a truncated SOCS3, generated by deleting the kinase inhibitory region (KIR) domain, failed to promote replication and translation of EV-D68. Based on the above studies, we designed a short peptide named SOCS3 inhibitor, which can specifically bind and inhibit the KIR structural domain of SOCS3, significantly reducing the RNA and protein levels of EV-D68. In summary, our results demonstrated a novel mechanism by which EV-D68 inhibits ISG transcription and antagonizes the antiviral responses of host type I interferon.

Estrogen receptor α-mediated signaling inhibits type I interferon response to promote breast carcinogenesis.

Estrogen receptor α (ERα)is an important driver and therapeutic target in ∼70% of breast cancers. How ERαdrives breast carcinogenesis is not fully understood. In this study, we show that ERαis a negative regulator of type I interferon (IFN) response. Activation of ERα by its natural ligand estradiol inhibits IFN-β-induced transcription of downstream IFN-stimulated genes (ISGs), whereas ERα deficiency or the stimulation with its antagonist fulvestrant has opposite effects. Mechanistically, ERα induces the expression of the histone 2A variant H2A.Z to restrict the engagement of the IFN-stimulated gene factor 3 (ISGF3) complex to the promoters of ISGs and also interacts with STAT2 to disrupt the assembly of the ISGF3 complex. These two events mutually lead to the inhibition of ISG transcription induced by type I IFNs. In a xenograft mouse model, fulvestrant enhances the ability of IFN-β to suppress ERα+ breast tumor growth. Consistently, clinical data analysis reveals that ERα+ breast cancer patients with higher levels of ISGs exhibit higher long-term survival rates. Taken together, our findings suggest that ERαinhibits type I IFN response via two distinct mechanisms to promote breast carcinogenesis.

Transcriptional changes in multiple endocrine organs from lethal cases of COVID-19

Altered circulating hormone and metabolite levels have been reported during and post-COVID-19. Yet, studies of gene expression at the tissue level capable of identifying the causes of endocrine dysfunctions are lacking. Transcript levels of endocrine-specific genes were analyzed in five endocrine organs of lethal COVID-19 cases. Overall, 116 autoptic specimens from 77 individuals (50 COVID-19 cases and 27 uninfected controls) were included. Samples were tested for the SARS-CoV-2 genome. The adrenals, pancreas, ovary, thyroid, and white adipose tissue (WAT) were investigated. Transcript levels of 42 endocrine-specific and 3 interferon-stimulated genes (ISGs) were measured and compared between COVID-19 cases (virus-positive and virus-negative in each tissue) and uninfected controls. ISG transcript levels were enhanced in SARS-CoV-2-positive tissues. Endocrine-specific genes (e.g., HSD3B2, INS, IAPP, TSHR, FOXE1, LEP, and CRYGD) were deregulated in COVID-19 cases in an organ-specific manner. Transcription of organ-specific genes was suppressed in virus-positive specimens of the ovary, pancreas, and thyroid but enhanced in the adrenals. In WAT of COVID-19 cases, transcription of ISGs and leptin was enhanced independently of virus detection in tissue. Though vaccination and prior infection have a protective role against acute and long-term effects of COVID-19, clinicians must be aware that endocrine manifestations can derive from virus-induced and/or stress-induced transcriptional changes of individual endocrine genes.Key messages• SARS-CoV-2 can infect adipose tissue, adrenals, ovary, pancreas and thyroid.• Infection of endocrine organs induces interferon response.• Interferon response is observed in adipose tissue independently of virus presence.• Endocrine-specific genes are deregulated in an organ-specific manner in COVID-19.• Transcription of crucial genes such as INS, TSHR and LEP is altered in COVID-19.

Interplay between PML NBs and HIRA for H3.3 dynamics following type I interferon stimulus.

Promyelocytic leukemia Nuclear Bodies (PML NBs) are nuclear membrane-less organelles physically associated with chromatin underscoring their crucial role in genome function. The H3.3 histone chaperone complex HIRA accumulates in PML NBs upon senescence, viral infection or IFN-I treatment in primary cells. Yet, the molecular mechanisms of this partitioning and its function in regulating histone dynamics have remained elusive. By using specific approaches, we identify intermolecular SUMO-SIM interactions as an essential mechanism for HIRA recruitment in PML NBs. Hence, we describe a role of PML NBs as nuclear depot centers to regulate HIRA distribution in the nucleus, dependent both on SP100 and DAXX/H3.3 levels. Upon IFN-I stimulation, PML is required for interferon-stimulated genes (ISGs) transcription and PML NBs become juxtaposed to ISGs loci at late time points of IFN-I treatment. HIRA and PML are necessary for the prolonged H3.3 deposition at the transcriptional end sites of ISGs, well beyond the peak of transcription. Though, HIRA accumulation in PML NBs is dispensable for H3.3 deposition on ISGs. We thus uncover a dual function for PML/PML NBs, as buffering centers modulating the nuclear distribution of HIRA, and as chromosomal hubs regulating ISGs transcription and thus HIRA-mediated H3.3 deposition at ISGs upon inflammatory response.

Monocytes differentiate along two alternative pathways during sterile inflammation.

During inflammation, monocytes differentiate within tissues into macrophages (mo-Mac) or dendritic cells (mo-DC). Whether these two populations derive from alternative differentiation pathways or represent different stages along a continuum remains unclear. Here, we address this question using temporal single-cell RNA sequencing in an in vitro model, allowing the simultaneous differentiation of human mo-Mac and mo-DC. We find divergent differentiation paths, with a fate decision occurring within the first 24 h and confirm this result in vivo using a mouse model of sterile peritonitis. Using a computational approach, we identify candidate transcription factors potentially involved in monocyte fate commitment. We demonstrate that IRF1 is necessary for mo-Mac differentiation, independently of its role in regulating transcription of interferon-stimulated genes. In addition, we describe the transcription factors ZNF366 and MAFF as regulators of mo-DC development. Our results indicate that mo-Macs and mo-DCs represent two alternative cell fates requiring distinct transcription factors for their differentiation.

Abstract 4418: Heterogeneity of IFN-γ responsiveness in myeloid malignancies- implication for the possible impact on IFN-γ immunotherapy

Abstract Introduction: Allogeneic stem cell transplantation (alloSCT) can potentially cure hematologic malignancies through graft-versus-leukemia effect (GVL). Several studies have suggested that IFN-γ pathway is critical for GVL sensitivity or resistance in leukemia cells. In mouse model, acute myeloid leukemia (AML) generated in IFN-γ receptor-deficient mice acquired resistance to GVL (Matte-Martone, Shlomchik WD, J Clin Invest. 2017). Other groups have reported that IFN-γ restores HLA expression on myeloid blasts from patients who relapsed AMLs after alloSCT. These data support the clinical application of IFN-γ to augment GVL in subjects with post-transplant relapse. However, AML is molecularly heterogeneous, and the IFN-γ responsiveness may vary by AML subtypes. We analyzed IFN-γ responsiveness of primary AML cells and investigated the role of IFN-γ on cell death and T-cell recognition of AML cell lines. Methods: Primary AML cells collected at diagnosis (n=60), at relapse after chemotherapy (n=4), and at post-transplant relapse (n=8) were stimulated with IFN-γ at 1 ng/ml or 50 ng/mL in vitro, and IFN-γ responsiveness was analyzed by measuring phosphorylation of STAT1 (pSTAT) after 15 minutes and upregulation of HLA-DR after 48 hours by flow cytometry. U937 and THP-1 cell lines were used to investigate IFN-γ induced cell death and the impact of IFN-γ priming of leukemia cells on the activation of alloreactive T cells. For this, Jurkat cells transduced with anti-HA1 T cell receptor (αHA1-TCR-T) were used, and the activation was analyzed through CD69 upregulation by flow cytometry. Results: IFN-γ responsiveness was heterogeneous in both pSTAT1 and HLA-DR upregulation of primary AML blasts. IFN-γ responsiveness also varied within the subpopulation of leukemic blasts within the sample at diagnosis and relapse, implying clonal evolution. Compared to THP-1, U937 cells fail to upregulate HLA-DR with IFN-γ despite appropriate pSTAT1 response to IFN-γ, suggesting silencing of downstream IFN-γ activation site (GAS) and transcription of IFN-stimulated genes (ISGs). IFN-γ directly induced cell death in THP-1 in vitro, while U937 was resistant to IFN-γ induced cell death. Priming of THP-1 with IFN-γ significantly improved the affinity of αHA1-TCR-T to peptide-stimulated THP-1. Conclusion: IFN-γ responsiveness of AML blasts was heterogeneous, and cell line data suggest an impact of IFN-γ sensitivity on IFN-γ mediated cell death and alloreactive T cell recognition. Future studies aim to understand the mechanisms underlying IFN-γ sensitivity or resistance in AML, which would help to design clinical trials by selecting the patients who would obtain maximal benefit from IFN-γ therapy in post-transplant relapse (NCT04628338). Citation Format: Kedwin Ventura, Iuliia Kovalenko, Jui-En Ray Lee, Biswas Neupane, Daniel Stapor, Sawa Ito. Heterogeneity of IFN-γ responsiveness in myeloid malignancies- implication for the possible impact on IFN-γ immunotherapy. [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2023; Part 1 (Regular and Invited Abstracts); 2023 Apr 14-19; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2023;83(7_Suppl):Abstract nr 4418.

The main protease of SARS-CoV-2 cleaves histone deacetylases and DCP1A, attenuating the immune defense of the interferon-stimulated genes

Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), which causes coronavirus disease 2019, constitutes an emerging human pathogen of zoonotic origin. A critical role in protecting the host against invading pathogens is carried out by interferon-stimulated genes (ISGs), the primary effectors of the type I interferon (IFN) response. All coronaviruses studied thus far have to first overcome the inhibitory effects of the IFN/ISG system before establishing efficient viral replication. However, whether SARS-CoV-2 evades IFN antiviral immunity by manipulating ISG activation remains to be elucidated. Here, we show that the SARS-CoV-2 main protease (Mpro) significantly suppresses the expression and transcription of downstream ISGs driven by IFN-stimulated response elements in a dose-dependent manner, and similar negative regulations were observed in two mammalian epithelial cell lines (simian Vero E6 and human A549). Our analysis shows that to inhibit the ISG production, Mpro cleaves histone deacetylases (HDACs) rather than directly targeting IFN signal transducers. Interestingly, Mpro also abolishes the activity of ISG effector mRNA-decapping enzyme 1a (DCP1A) by cleaving it at residue Q343. In addition, Mpro from different genera of coronaviruses has the protease activity to cleave both HDAC2 and DCP1A, even though the alphacoronaviruse Mpro exhibits weaker catalytic activity in cleaving HDAC2. In conclusion, our findings clearly demonstrate that SARS-CoV-2 Mpro constitutes a critical anti-immune effector that modulates the IFN/ISG system at multiple levels, thus providing a novel molecular explanation for viral immune evasion and allowing for new therapeutic approaches against coronavirus disease 2019 infection.

Duck cGAS inhibits DNA and RNA virus replication by activating IFNs and antiviral ISGs.

Cyclic GMP-AMP Synthase (cGAS) is a pivotal adaptor of the signaling pathways involving the pattern recognition receptors and plays an important role in apoptosis and immune regulation. The cGAS function in mammals has been investigated extensively; however, the function of duck cGAS (du-cGAS) in response to viral infections is still unclear. This study aimed to clone the mallard (Anas platyrhynchos) cGAS homolog to investigate the function of duck cGAS (du-cGAS) in host antiviral innate immunity. The results showed that the open reading frame (ORF) region of the du-cGAS gene was 1296 bp, encoding 432 amino acids (aa) and exhibiting similar functional domains with its chicken counterpart. Knockdown of the endogenous du-cGAS by specific sgRNA strongly increased the replication of DNA viruses, including duck adenovirus B2 (DAdV B2) and duck short beak and dwarfism syndrome virus (SBDSV). However, the knockout did not impair the replication of novel duck reovirus (NDRV), an RNA virus. Furthermore, the mRNA expressions of type I interferon (IFNs) and vital interferon-stimulated genes (ISGs) were remarkably reduced in the du-cGAS knockout DEF cell line. Inversely, du-cGAS overexpression greatly activated the transcription of IFN-α, IFN-β, and vital ISGs, and impaired the replication of DAdV B2, SBDSV, and NDRV in the DEF cell line. Importantly, we found that a deletion of 68 aa in the N terminus didn't impair the antiviral function of du-cGAS. Overexpressing NTase Core, C-Domain (Mab21), or Zinc-Ribbon domain independently had no antiviral effects. Generally, these results reveal that du-cGAS is a vital component of the innate immune system of ducks, with a universal antiviral activity, and provides a useful strategy for the control of waterfowl viral diseases.

Innate immune activation without immune cell infiltration in brains of murine models of Aicardi-Goutières Syndrome.

Chronic inflammation is frequently invoked as a mechanism of neurodegeneration and yet inflammatory cell infiltrates are seldom seen in brains of these disorders. Different disciplines utilize different technologies and methodologies to describe what is immunologically defined as the innate immune response (IIR). We examined murine models of the human neurodegenerative disease Aicardi-Goutières Syndrome, where an IIR is initiated by aberrant RNA metabolism secondary to a mutation in adenosine deaminase acting on RNA gene (ADAR1). We previously showed that these mice demonstrated a deficit in RNA editing that lead to MDA-5 mediated RNA sensing pathway activation of the IIR with massive interferon stimulated gene transcription and translation. As early as 2 weeks of age, in situ hybridization demonstrated that different central nervous system (CNS) cell lineages expressed very high levels of distinct interferon stimulated genes (ISGs) in the absence of interferon and absence of immune cell infiltrates. We have expanded these studies to more completely describe the breadth of ISG expression systemically and in CNS using double label in situ hybridization. Within the CNS aberrant ISG expression was mostly limited to neurons, microglia, ependyma, choroid plexus, and endothelial cells with little expression in oligodendroglia and astrocytes except for STAT1. Wild type controls showed a similar pattern of ISG expression but only in aged mice and at levels minimally detectable by in situ hybridization. Despite months of elevated ISG expression in mutant mice, there was essentially no inflammatory infiltrate, no interferon production and minimal glial reaction. Histomorphological neurodegenerative pathology of ventricular dilatation and deep gray matter mineralization were evident in mutant mice 8-13 months of age but this did not show a spatial relationship to ISG expression. This IIR without immune cell infiltration leads to neurodegeneration through non-canonical pathways that may accentuate normal aging pathways.

Toll-like receptor 3 signaling induces interferon-induced transmembrane protein 1 in BEAS-2B cells.

The human bronchial epithelium plays a crucial role in mediating antiviral immune reactions. When double-stranded RNA (dsRNA) binds to the receptor named Toll-like receptor (TLR) 3, activation of antiviral innate immune reactions is initiated by producing interferon (IFN) type I. Then, type I IFN promotes the transcription of IFN-stimulated genes (ISGs). Proteins encoded by ISGs reveal antiviral effects. The IFN-induced transmembrane protein 1 (IFITM1) is an ISG family member that inhibits viral infection by preventing the entry of viruses with a cell membrane. However, IFITM1 expression in human bronchial epithelium remains largely undetermined. Here, we investigated whether IFITM1 is expressed in cultured BEAS-2B bronchial epithelial cells. Polyinosinic:polycytidylic acid (poly I:C) was used for treatment of BEAS-2B as a TLR3 ligand. IFITM1 expression levels were measured using reverse transcription-quantitative PCR and Western blotting. Using RNA interference, we determined the significance of IFN-β and ISG56 on IFITM1 upregulation. Poly I:C treatment significantly upregulated IFITM1 expression in BEAS-2B cells, and it was concentration- and time-dependent. Knockdown of IFN-β or ISG56 decreased poly I:C-induced IFITM1 expression levels. Recombinant IFN-β also increased expression levels of IFITM1. In BEAS-2B cells, IFITM1 expression is upregulated by poly I:C, at least partly, via the TLR3/IFN-β/ISG56 axis. Thus, IFITM1 may contribute to antiviral innate immunity in bronchial epithelium.

The chromatin remodeling protein ATRX positively regulates IRF3-dependent type I interferon production and interferon-induced gene expression.

The chromatin remodeling protein alpha thalassemia/mental retardation syndrome X-linked (ATRX) is a component of promyelocytic leukemia nuclear bodies (PML-NBs) and thereby mediates intrinsic immunity against several viruses including human cytomegalovirus (HCMV). As a consequence, viruses have evolved different mechanisms to antagonize ATRX, such as displacement from PML-NBs or degradation. Here, we show that depletion of ATRX results in an overall impaired antiviral state by decreasing transcription and subsequent secretion of type I IFNs, which is followed by reduced expression of interferon-stimulated genes (ISGs). ATRX interacts with the transcription factor interferon regulatory factor 3 (IRF3) and associates with the IFN-β promoter to facilitate transcription. Furthermore, whole transcriptome sequencing revealed that ATRX is required for efficient IFN-induced expression of a distinct set of ISGs. Mechanistically, we found that ATRX positively modulates chromatin accessibility specifically upon IFN signaling, thereby affecting promoter regions with recognition motifs for AP-1 family transcription factors. In summary, our study uncovers a novel co-activating function of the chromatin remodeling factor ATRX in innate immunity that regulates chromatin accessibility and subsequent transcription of interferons and ISGs. Consequently, ATRX antagonization by viral proteins and ATRX mutations in tumors represent important strategies to broadly compromise both intrinsic and innate immune responses.

E3 ubiquitin ligase NEURL3 promotes innate antiviral response through catalyzing K63-linked ubiquitination of IRF7.

Interferon regulatory factor 7 (IRF7), as the interferon-stimulated gene, maximally drives type I interferon (IFN) production. However, the mechanisms by which the biological function of IRF7 is regulated remain elusive. In this study, we found that IRF7 selectively interacted with the neuralized E3 ubiquitin-protein ligase 3 (NEURL3). In concomitant with IRF7 induction, NEURL3 is upregulated by NF-κB signaling in the late phase of viral infection. Moreover, NEURL3 augmented the host antiviral immune response through ubiquitinating IRF7. A mechanistic study revealed that NEURL3 triggered K63-linked poly-ubiquitination on IRF7 lysine 375, which in turn epigenetically enhanced the transcription of interferon-stimulated genes (ISGs) through disruption of the association of IRF7 with Histone Deacetylase 1 (HDAC1), consequently augmenting host antiviral immune response. Accordingly, Neurl3-/- mice produced less type I IFNs and exhibited increased susceptibility to viral infection. Taken together, our findings identify NEURL3 as an E3 ubiquitin ligase of IRF7 and shed new light on the positive regulation of IRF7 in host antiviral immune signaling.

Interactome of Aiolos/Ikaros Reveals Combination Rationale of Cereblon Modulators with HDAC Inhibitors in DLBCL.

Cereblon (CRBN), a substrate receptor of the E3 ubiquitin ligase complex CRL4CRBN, is the target of the small molecules lenalidomide and avadomide. Upon binding of the drugs, Aiolos and Ikaros are recruited to the E3 ligase, ubiquitylated, and subsequently degraded. In diffuse large B-cell lymphoma (DLBCL) cells, Aiolos and Ikaros are direct transcriptional repressors of interferon-stimulated genes (ISG) and degradation of these substrates results in increased ISG protein levels resulting in decreased proliferation and apoptosis. Herein, we aimed to uncover the mechanism(s) Aiolos and Ikaros use to repress ISG transcription and provide a mechanistic rationale for a combination strategy to enhance cell autonomous activities of CRBN modulators (CELMoD). We conducted paired RNA sequencing with histone modification and Aiolos/Ikaros chromatin immunoprecipitation sequencing to identify genes regulated by these transcription factors and to elucidate correlations to drug sensitivity. We confirmed Aiolos/Ikaros mediated transcriptional complex formation in DLBCL patient samples including those treated with avadomide. In DLBCL, the repression of ISG transcription is accomplished in part through recruitment of large transcriptional complexes such as the nucleosome remodeling and deacetylase, which modify the chromatin landscape of these promoters. A rational combination approach of avadomide with a specific histone deacetylase inhibitor leads to a significant increase in ISG transcription compared with either single agent, and synergistic antiproliferative activity in DLBCL cell lines. Our results provide a novel role for lineage factors Aiolos and Ikaros in DLBCL as well as further insight into the mechanism(s) of Aiolos and Ikaros-mediated transcriptional repression and unique therapeutic combination strategies.