IFN-β mRNA Research Articles

Lactylation of RNA m6A demethylase ALKBH5 promotes innate immune response to DNA herpesviruses and mpox virus

RNA N6-methyladenosine (m6A) demethylase AlkB homolog 5 (ALKBH5) plays a crucial role in regulating innate immunity. Lysine acylation, a widespread protein modification, influences protein function, but its impact on ALKBH5 during viral infections has not been well characterized. This study investigates the presence and regulatory mechanisms of a previously unidentified lysine acylation in ALKBH5 and its role in mediating m6A modifications to activate antiviral innate immune responses. We demonstrate that ALKBH5 undergoes lactylation, which is essential for an effective innate immune response against DNA herpesviruses, including herpes simplex virus type 1 (HSV-1), Kaposi's sarcoma-associated herpesvirus (KSHV), and mpox virus (MPXV). This lactylation attenuates viral replication. Mechanistically, viral infections enhance ALKBH5 lactylation by increasing its interaction with acetyltransferase ESCO2 and decreasing its interaction with deacetyltransferase SIRT6. Lactylated ALKBH5 binds interferon-beta (IFN-β) messenger RNA (mRNA), leading to demethylation of its m6A modifications and promoting IFN-β mRNA biogenesis. Overexpression of ESCO2 or depletion of SIRT6 further enhances ALKBH5 lactylation to strengthen IFN-β mRNA biogenesis. Our results identify a posttranslational modification of ALKBH5 and its role in regulating antiviral innate immune responses through m6A modification. The finding provides an understanding of innate immunity and offers a potential therapeutic target for HSV-1, KSHV, and MPXV infections.

E3 Ubiquitin Ligase Smurf1 Regulates the Inflammatory Response in Macrophages and Attenuates Hepatic Damage during Betacoronavirus Infection.

The E3 ubiquitin ligase Smurf1 catalyzes the ubiquitination and proteasomal degradation of several protein substrates related to inflammatory responses and antiviral signaling. This study investigated the role of Smurf1 in modulating inflammation induced by Betacoronavirus infection. Bone marrow-derived macrophages (BMDMs) from C57BL/6 (wild-type) or Smurf1-deficient (Smurf1-/-) mice were infected with MHV-A59 to evaluate the inflammatory response in vitro. Smurf1 was found to be required to downregulate the macrophage production of pro-inflammatory mediators, including TNF, and CXCL1; to control viral release from infected cells; and to increase cell viability. To assess the impact of Smurf 1 in vivo, we evaluated the infection of mice with MHV-A59 through the intranasal route. Smurf1-/- mice infected with a lethal inoculum of MHV-A59 succumbed earlier to infection. Intranasal inoculation with a 10-fold lower dose of MHV-A59 resulted in hematological parameter alterations in Smurf1-/- mice suggestive of exacerbated systemic inflammation. In the lung parenchyma, Smurf1 expression was essential to promote viral clearance, downregulating IFN-β mRNA and controlling the inflammatory profile of macrophages and neutrophils. Conversely, Smurf1 did not affect IFN-β mRNA regulation in the liver, but it was required to increase TNF and iNOS expression in neutrophils and decrease TNF expression in macrophages. In addition, Smurf1-/- mice exhibited augmented liver injuries, accompanied by high serum levels of alanine aminotransferase (ALT). These findings suggest that Smurf1 plays a critical role in regulating the inflammatory response in macrophages and attenuating systemic inflammation during Betacoronavirus infection.

The role of 2ʹ–5ʹ-oligoadenylate synthase-like protein (OASL1) in biliary and hepatotoxin-induced liver injury in mice

Following an injury, the liver embarks on a process that drives the accumulation and reformation of the extracellular matrix, leading to hepatic fibrosis. Type I interferons (IFNs), including IFN-α and IFN-β, play a crucial role in averting chronic liver injury through the activation of IFN-stimulated genes (ISGs), which are instrumental in sculpting adaptive immunity. The role of 2ʹ–5ʹ-oligoadenylate synthase-like protein 1 (OASL1), an antiviral ISG, in the context of liver fibrosis remains to be elucidated. To elicit liver fibrosis, a diet containing 0.1% diethoxycarbonyl-1,4-dihydrocollidine (DDC) and carbon tetrachloride (CCl4) were employed to induce cholestatic- and hepatotoxin-mediated liver fibrosis, respectively. Histological analyses of both models revealed that OASL1−/− mice exhibited reduced liver damage and, consequently, expressed lower levels of fibrotic mediators, notably α-smooth muscle actin. OASL1−/− mice demonstrated significantly elevated IFN-α and IFN-β mRNA levels, regulated by the IFN regulatory factor 7 (IRF7). Additionally, OASL1−/− ameliorated chronic liver fibrosis through the modulation of nuclear factor-κB (NF-κB) signaling. The effect of OASL1 on type I IFN production in acute liver damage was further explored and OASL1−/− mice consistently showed lower alanine transaminase levels and pro-inflammatory cytokines, but IFN-α and IFN-β mRNA levels were upregulated, leading to amelioration of acute liver injury. Additionally, the study discovered that F4/80-positive cells were observed more frequently in OASL1−/− CCl4 acutely treated mice. This implies that there is a significant synergy in the function of macrophages and OASL1 deficiency. These results demonstrate that in instances of liver injury, OASL1 inhibits the production of type I IFN by modulating the NF-κB signaling pathway, thereby worsening disease.

Semen Strychni Pulveratum and vomicine alleviate neuroinflammation in amyotrophic lateral sclerosis through cGAS-STING-TBK1 pathway

Ethnopharmacological relevanceAmyotrophic lateral sclerosis (ALS) is a fetal neuromuscular disorder characterized by the gradual deterioration of motor neurons. Semen Strychni pulveratum (SSP), a processed version of Semen Strychni (SS) powder, is widely used to treat ALS in China. Vomicine is one of the most primary components of SS. However, their pharmacological effects and mechanisms for ALS remain elusive. Aim of the studyThis study aimed to evaluate the neuroprotective and anti-neuroinflammatory effects of SSP and vomicine, as well as to explore their protective roles in ALS and the underlying mechanisms. Materials and methodsIn vivo, 8-week-old hSOD1-WT mice and hSOD1-G93A mice were orally administered different concentrations of SSP (SSP-L = 5.46 mg/ml, SSP-M = 10.92 mg/ml or SSP-H = 16.38 mg/ml) once every other day for 8 weeks. A series of experiments, including body weight measurement, footprint tests, Hematoxylin & Eosin staining, and Nissl staining, were performed to evaluate the preventive effect of SSP. Immunofluorescence staining, western blotting, and RT-qPCR were subsequently performed to evaluate activation of the cGAS-STING-TBK1 pathway in the spinal cord. In vitro, hSOD1G93A NSC-34 cells were treated with vomicine to further explore the pharmacological mechanism of vomicine in the treatment of ALS via the cGAS-STING-TBK1 pathway. ResultsSSP improved motor function, body weight loss, gastrocnemius muscle atrophy, and motor neuron loss in the spine and cortex of hSOD1-G93A mice. Furthermore, the cGAS-STING-TBK1 pathway was activated in the spinal cord of hSOD1-G93A mice, with activation predominantly observed in neurons and microglia. However, the levels of cGAS, STING, and pTBK1 proteins and cGAS, IRF3, IL-6, and IL-1β mRNA were reversed following intervention with SSP. Vomicine not only downregulated the levels of cGAS, TBK1, IL-6 and IFN-β mRNA, but also the levels of cGAS and STING protein in hSOD1G93A NSC-34 cells. ConclusionThis study demonstrated that SSP and vomicine exert neuroprotective and anti-neuroinflammatory effects in the treatment of ALS. SSP and vomicine may reduce neuroinflammation by regulating the cGAS-STING-TBK1 pathway, and could thereby play a role in ALS treatment.

CD11b maintains West Nile virus replication through modulation of immune response in human neuroblastoma cells

BackgroundWest Nile virus (WNV) is a rapidly spreading mosquito-borne virus accounted for neuroinvasive diseases. An insight into WNV-host factors interaction is necessary for development of therapeutic approaches against WNV infection. CD11b has key biological functions and been identified as a therapeutic target for several human diseases. The purpose of this study was to determine whether CD11b was implicated in WNV infection.MethodsSH-SY5Y cells with and without MEK1/2 inhibitor U0126 or AKT inhibitor MK-2206 treatment were infected with WNV. CD11b mRNA levels were assessed by real-time PCR. WNV replication and expression of stress (ATF6 and CHOP), pro-inflammatory (TNF-α), and antiviral (IFN-α, IFN-β, and IFN-γ) factors were evaluated in WNV-infected SH-SY5Y cells with CD11b siRNA transfection. Cell viability was determined by MTS assay.ResultsCD11b mRNA expression was remarkably up-regulated by WNV in a time-dependent manner. U0126 but not MK-2206 treatment reduced the CD11b induction by WNV. CD11b knockdown significantly decreased WNV replication and protected the infected cells. CD11b knockdown markedly increased TNF-α, IFN-α, IFN-β, and IFN-γ mRNA expression induced by WNV. ATF6 mRNA expression was reduced upon CD11b knockdown following WNV infection.ConclusionThese results demonstrate that CD11b is involved in maintaining WNV replication and modulating inflammatory as well as antiviral immune response, highlighting the potential of CD11b as a target for therapeutics for WNV infection.

NAD activates olfactory receptor 1386 to regulate type I interferon responses in Plasmodium yoelii YM infection

Olfactory receptors (Olfr) are G protein-coupled receptors that are normally expressed on olfactory sensory neurons to detect volatile chemicals or odorants. Interestingly, many Olfrs are also expressed in diverse tissues and function in cell-cell recognition, migration, and proliferation as well as immune responses and disease processes. Here, we showed that many Olfr genes were expressed in the mouse spleen, linked to Plasmodium yoelii genetic loci significantly, and/or had genome-wide patterns of LOD scores (GPLSs) similar to those of host Toll-like receptor genes. Expression of specific Olfr genes such as Olfr1386 in HEK293T cells significantly increased luciferase signals driven by IFN-β and NF-κB promoters, with elevated levels of phosphorylated TBK1, IRF3, P38, and JNK. Mice without Olfr1386 were generated using the CRISPR/Cas9 method, and the Olfr1386-/- mice showed significantly lower IFN-α/β levels and longer survival than wild-type (WT) littermates after infection with P. yoelii YM parasites. Inhibition of G protein signaling and P38 activity could affect cyclic AMP-responsive element promoter-driven luciferase signals and IFN-β mRNA levels in HEK293T cells expressing the Olfr1386 gene, respectively. Screening of malaria parasite metabolites identified nicotinamide adenine dinucleotide (NAD) as a potential ligand for Olfr1386, and NAD could stimulate IFN-β responses and phosphorylation of TBK1 and STAT1/2 in RAW264.7 cells. Additionally, parasite RNA (pRNA) could significantly increase Olfr1386 mRNA levels. This study links multiple Olfrs to host immune response pathways, identifies a candidate ligand for Olfr1386, and demonstrates the important roles of Olfr1386 in regulating type I interferon (IFN-I) responses during malaria parasite infections.

TLR4/7-mediated host-defense responses of gingival epithelial cells.

Gingival epithelial cells (GECs) are physical and immunological barriers against outward pathogens while coping with a plethora of non-pathogenic commensal bacteria. GECs express several members of Toll-like receptors (TLRs) and control subsequent innate immune responses. TLR4 senses lipopolysaccharide (LPS) while TLR7/8 recognizes single-strand RNA (ssRNA) playing important roles against viral infection. However, their distinct roles in GECs have not been fully demonstrated. Here, we analyzed biological responses of GECs to LPSand CL075, a TLR7/8 agonist. GE1, a mouse gingival epithelial cell line, constitutively express TLR4 and TLR7, but not TLR8, like primary skin keratinocytes. Stimulation of GE1 cells with CL075 induced cytokine, chemokine, and antimicrobial peptide expressions, the pattern of which is rather different from that with LPS: higher mRNA levels of interferon (IFN) β, CXCL10, and β-defensin (BD) 14 (mouse homolog of human BD3); lower levels of tumor necrosis factor (TNF), CCL5, CCL11, CCL20, CXCL2, and CX3CL1. As for the intracellular signal transduction of GE1 cells, CL075 rapidly induced significant AKT phosphorylation but failed to activate IKKα/β-NFκB pathway, whereas LPS induced marked IKKα/β-NFκB activation without significant AKT phosphorylation. In contrast, both CL075 and LPS induced rapid IKKα/β-NFκB activation and AKT phosphorylation in a macrophage cell line. Furthermore, specific inhibition of AKT activity abrogated CL075-induced IFNβ, CXCL10, and BD14 mRNA expression in GE1 cells. Thus, TLR4/7 ligands appear to induce rather different host-defense responses of GECs through distinct intracellular signaling mechanisms.

Dampening of ISGylation of RIG-I by ADAP regulates type I interferon response of macrophages to RNA virus infection.

While macrophage is one of the major type I interferon (IFN-I) producers in multiple tissues during viral infections, it also serves as an important target cell for many RNA viruses. However, the regulatory mechanism for the IFN-I response of macrophages to respond to a viral challenge is not fully understood. Here we report ADAP, an immune adaptor protein, is indispensable for the induction of the IFN-I response of macrophages to RNA virus infections via an inhibition of the conjugation of ubiquitin-like ISG15 (ISGylation) to RIG-I. Loss of ADAP increases RNA virus replication in macrophages, accompanied with a decrease in LPS-induced IFN-β and ISG15 mRNA expression and an impairment in the RNA virus-induced phosphorylation of IRF3 and TBK1. Moreover, using Adap-/- mice, we show ADAP deficiency strongly increases the susceptibility of macrophages to RNA-virus infection in vivo. Mechanically, ADAP selectively interacts and functionally cooperates with RIG-I but not MDA5 in the activation of IFN-β transcription. Loss of ADAP results in an enhancement of ISGylation of RIG-I, whereas overexpression of ADAP exhibits the opposite effect in vitro, indicating ADAP is detrimental to the RNA virus-induced ISGylation of RIG-I. Together, our data demonstrate a novel antagonistic activity of ADAP in the cell-intrinsic control of RIG-I ISGylation, which is indispensable for initiating and sustaining the IFN-I response of macrophages to RNA virus infections and replication.

Effects of toll-like receptor agonists and SARS-CoV-2 antigens on interferon (IFN) expression by peripheral blood CD3+ T cells from COVID-19 patients

BackgroundSignaling by toll-like receptors (TLRs) initiates important immune responses against viral infection. The role of TLRs in severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) infection is not well elucidated. Thus, we investigated the interaction of TLRs agonists and SARS-COV-2 antigens with immune cells in vitro. Material & methods30 coronavirus disease 2019 (COVID-19) patients (15 severe and 15 moderate) and 10 age and sex-matched healthy control (HC) were enrolled. Peripheral blood mononuclear cells (PBMCs) were isolated and activated with TLR3, 7, 8, and 9 agonists, the spike protein (SP) of SARS-CoV-2, and the receptor binding domain (RBD) of SP. Frequencies of CD3+IFN-β+ T cells, and CD3+IFN-γ+ T cells were evaluated by flow cytometry. Interferon (IFN)-β gene expression was assessed by qRT-PCR. ResultsThe frequency of CD3+IFN-β+ T cells was higher in PBMCs from moderate (p < 0.0001) and severe (p = 0.009) patients at baseline in comparison with HCs. The highest increase in the frequency of CD3+IFN-β+ T cells in cell from moderate patients was induced by TLR8 agonist and SP (p < 0.0001 for both) when compared to HC, while, the highest increase of the frequency of CD3+IFN-β+ T cells in sample of severe patients was seen with TLR8 and TLR7 agonists (both p = 0.002). The frequency of CD3+IFN-γ+ T cells was significantly increased upon stimulation with TLR agonists in cell from patients with moderate and severe COVID-19, compared with HC (all p < 0.01), except with TLR7 and TLR8 agonists. The TLR8 agonist did not significantly increase the frequency of CD3+IFN-γ+ T cells in PBMCs of severe patients, but did so in cells from patients with moderate disease (p = 0.01). Moreover, IFN-β gene expression was significantly upregulated in CD3+T cells from moderate (p < 0.0001) and severe (p = 0.002) COVID-19 patients, compared to HC after stimulation with the TLR8 agonist, while, stimulation of T cells with SP, significantly up-regulated IFN-β mRNA expression in cells from patients with moderate (p = 0.0003), but not severe disease. ConclusionStimulation of PBMCs from COVID-19 patients, especially patients with moderate disease, with TLR8 agonist and SP increased the frequency of IFN-β-producing T cells and IFN-β gene expression.

Mammalian reovirus µ1 protein attenuates RIG-I and MDA5-mediated signaling transduction by blocking IRF3 phosphorylation and nuclear translocation

Mammalian reovirus (MRV) is a non-enveloped, gene segmented double-stranded RNA (dsRNA) virus. It is an important zoonotic pathogen that infects many mammals and vertebrates that act as natural hosts and causes respiratory and digestive tract diseases. Studies have reported that RIG-I and MDA5 in the innate immune cytoplasmic RNA-sensing RIG-like receptor (RLR) signaling pathway can recognize dsRNA from MRV and promote antiviral type I interferon (IFN) responses. However, the mechanism by which many MRV-encoded proteins evade the host innate immune response remains unclear. Here, we show that exogenous μ1 protein promoted the proliferation of MRV in vitro, while knockdown of MRV μ1 protein expression by shRNA could impair MRV proliferation. Specifically, μ1 protein inhibited MRV or poly(I:C)-induced IFN-β expression, and attenuated RIG-I/MDA5-mediated signaling axis transduction during MRV infection. Importantly, we found that μ1 protein significantly decreased IFN-β mRNA expression induced by MDA5, RIG-I, MAVS, TBK1, IRF3(5D), and degraded the protein expression of exogenous MDA5, RIG-I, MAVS, TBK1 and IRF3 via the proteasomal and lysosomal pathways. Additionally, we show that μ1 protein can physically interact with MDA5, RIG-I, MAVS, TBK1, and IRF3 and attenuate the RIG-I/MDA5-mediated signaling cascades by blocking the phosphorylation and nuclear translocation of IRF3. In conclusion, our findings reveal that MRV outer capsid protein μ1 is a key factor in antagonizing RLRs signaling cascades and provide new strategies for effective prevention and treatment of MRV infection.

Oligoadenylate synthetase 1 displays dual antiviral mechanisms in driving translational shutdown and protecting interferon production

In response to viral infection, how cells balance translational shutdown to limit viral replication and the induction of antiviral components like interferons (IFNs) is not well understood. Moreover, how distinct isoforms of IFN-induced oligoadenylate synthetase 1 (OAS1) contribute to this antiviral response also requires further elucidation. Here, we show that human, but not mouse, OAS1 inhibits SARS-CoV-2 replication through its canonical enzyme activity via RNase L. In contrast, both mouse and human OAS1 protect against West Nile virus infection by a mechanism distinct from canonical RNase L activation. OAS1 binds AU-rich elements (AREs) of specific mRNAs, including IFNβ. This binding leads to the sequestration of IFNβ mRNA to the endomembrane regions, resulting in prolonged half-life and continued translation. Thus, OAS1 is an ARE-binding protein with two mechanisms of antiviral activity: driving inhibition of translation but also a broader, non-canonical function of protecting IFN expression from translational shutdown.

A245 CROHN’S DISEASE-ASSOCIATED BACTERIAL PATHOBIONT EVOKED EPITHELIAL MITOCHONDRIAL DAMAGE DRIVES TYPE I INTERFERON EXPRESSION

Abstract Background The Crohn’s disease-associated adherent invasive E. coli (AIEC) (strain LF82) decreases mitochondrial membrane potential and fragments the epithelial mitochondrial network. Due to the evolutionary history of mitochondria as former bacteria, they present a variety of molecular patterns known to trigger innate immune responses. Specifically, mitochondrial DNA (mtDNA) released following mitochondrial damage causes upregulation of type I interferons, priming inflammatory signalling. Thus, we hypothesized that AIEC-induced mitochondrial dysfunction results in mtDNA release to activate innate immune signalling that could contribute to disease pathogenesis in the gut. Aims We aimed determine if an inflammatory response occurs downstream of mitochondrial damage evoked by E. coli-LF82 infection. Methods Control T84 epithelial cells, T84s infected E. coli-LF82 (108 cfu, MOI=100, 4h) and T84s infected with the commensal E. coli-HB101 (108 cfu for an MOI=100, 4h) were collected for protein and RNA extraction. RNA was used to measure IFNα and IFNβ expression following infection, while protein was used to measure upstream mediators including phosphorylation of TANK Binding Kinase-1 (TBK1) via Western blot. Results Epithelia infected with E. coli-LF82 but not E. coli-HB101 displayed dramatic fragmentation of their mitochondria network and increased levels of phosphorylated-TBK1 compared to control T84 cells (pampersand:003C0.01). Assessment of IFNα and IFNβ mRNA revealed significant up-regulation E. coli-LF82 infected T84 cells and human colon organoids. Conclusions Our findings identify a novel role for E. coli-LF82 in activating type I interferon response and for the first time show AIEC-induced activation of TBK1 and type I interferons, which may occur through mitochondrial damage, rather than through response to bacterial molecular patterns. As a result, our findings suggest that the AIEC colonization seen in Crohn’s disease may dysregulate inflammatory signalling through mitochondrial damage. Ultimately, we believe this dysregulation of inflammatory signalling may sensitize the host to further inflammatory signalling, contributing to the pathogenesis of inflammatory bowel disease. Funding Agencies CIHR

Differential type I and type III interferon expression profiles in rheumatoid and juvenile idiopathic arthritis.

The role of type I and type III interferons (IFNs) in rheumatoid arthritis (RA) and juvenile idiopathic arthritis (JIA) is still poorly understood. The objective of this study was to examine the hypothesis that IFN expression profiles in the peripheral blood differ between subsets of arthritic subjects. Multiple type I and type III IFNs were examined in patients with RA and JIA, as well as among subtypes of JIA. Treatment-naïve RA and JIA patients were enrolled. Droplet digital PCR was used to measure the expression of type I, II, and III interferons in blood and synovial fluid leukocytes. Dendritic cell subsets were isolated from synovial fluid to examine IFN expression in each subset. Additionally, synovial mononuclear cells and JIA-derived fibroblast-like synoviocytes were stimulated with TNF, IFNγ, and poly(I:C) to examine inducible IFN expression. The predominant type I IFN gene expressed by blood leukocytes was IFNκ and was significantly lower in RA than JIA and controls. Oligoarticular and psoriatic JIA subgroups showed higher IFNκ expression compared to polyarticular JIA and RA. JIA synovial fluid leukocytes expressed abundant IFNγ and type III IFNs (IFNλ1, IFNλ3), with distinct dendritic cell subset contributions. JIA fibroblast-like synoviocytes produced IFNβ, IFNλ1, and IFNλ2 mRNA upon poly(I:C) stimulation. This study revealed differences in IFN expression patterns in RA and JIA, with notable differences between JIA subtypes. The expression levels of IFNκ, IFNγ, IFNλ1 and IFNλ3 in JIA suggest specific roles in disease pathology, influenced by disease subtype and joint microenvironment. This study contributes to understanding IFN-mediated mechanisms in arthritis, potentially guiding targeted therapeutic strategies.

Multiple unfolded protein response pathways cooperate to link cytosolic dsDNA release to stimulator of interferon gene activation.

The double-stranded DNA (dsDNA) sensor STING has been increasingly implicated in responses to "sterile" endogenous threats and pathogens without nominal DNA or cyclic di-nucleotide stimuli. Previous work showed an endoplasmic reticulum (ER) stress response, known as the unfolded protein response (UPR), activates STING. Herein, we sought to determine if ER stress generated a STING ligand, and to identify the UPR pathways involved. Induction of IFN-β expression following stimulation with the UPR inducer thapsigargin (TPG) or oxygen glucose deprivation required both STING and the dsDNA-sensing cyclic GMP-AMP synthase (cGAS). Furthermore, TPG increased cytosolic mitochondrial DNA, and immunofluorescence visualized dsDNA punctae in murine and human cells, providing a cGAS stimulus. N-acetylcysteine decreased IFN-β induction by TPG, implicating reactive oxygen species (ROS). However, mitoTEMPO, a mitochondrial oxidative stress inhibitor did not impact TPG-induced IFN. On the other hand, inhibiting the inositol requiring enzyme 1 (IRE1) ER stress sensor and its target transcription factor XBP1 decreased the generation of cytosolic dsDNA. iNOS upregulation was XBP1-dependent, and an iNOS inhibitor decreased cytosolic dsDNA and IFN-β, implicating ROS downstream of the IRE1-XBP1 pathway. Inhibition of the PKR-like ER kinase (PERK) pathway also attenuated cytoplasmic dsDNA release. The PERK-regulated apoptotic factor Bim was required for both dsDNA release and IFN-β mRNA induction. Finally, XBP1 and PERK pathways contributed to cytosolic dsDNA release and IFN-induction by the RNA virus, Vesicular Stomatitis Virus (VSV). Together, our findings suggest that ER stressors, including viral pathogens without nominal STING or cGAS ligands such as RNA viruses, trigger multiple canonical UPR pathways that cooperate to activate STING and downstream IFN-β via mitochondrial dsDNA release.

Luteolin-7-O-glucoside promotes macrophage release of IFN-β by maintaining mitochondrial function and corrects the disorder of glucose metabolism during RSV infection

Respiratory syncytial virus (RSV) pneumonia is the main cause of acute bronchiolitis in infants. Luteolin-7-O-glucoside (LUT-7G) is a natural flavonoid, which exists in a variety of plants and has the potential to treat viral pneumonia. We established RSV pneumonia mouse models and RSV-infected cell models. Clodronate liposomes were used to deplete macrophages. We used HE staining and immunohistochemistry to determine inflammatory damage and virus replication. We detected the expression levels of inflammatory factors and IFN-β through qPCR and ELISA. JC-1 kit was used for detecting the cell mitochondrial Membrane potential (MMP). ROS, SOD, and MDA kits were used for detecting intracellular oxidative stress damage. Metabolites of TCA in lung tissue and serum of mice were detected by GC-MS. Pharmacodynamic studies have shown that intervention with LUT-7G can alleviate lung tissue damage caused by RSV infection, inhibit RSV replication, and downregulate TNF-α, IL-1β, and IL-6 mRNA expression. LUT-7G upregulated the IFN-β content and the expression of IFN-β, ISG15, and OAS1 mRNA. In vitro, LUT-7G inhibited RSV-induced cell death, reversed the RSV-induced decrease of MMP and decreased intracellular oxidative stress. Target metabonomics showed that RSV infection upregulated the levels of glycolysis and TCA metabolites in lung tissue and serum, while LUT-7G could improve the disorder of glucose metabolism. The results indicate that LUT-7G can promote the release of IFN-β in the lung, alleviate inflammatory damage, and inhibit RSV replication during RSV infection. These effects may be achieved by protecting the mitochondrial function of alveolar macrophages and correcting the disorder of glucose metabolism.

4-Octyl itaconate inhibits poly(I:C)-induced interferon-β secretion in mouse bone marrow-derived macrophages partially by activating Nrf2

Viruses have become a major threat to human health. Interferon-β (IFN-β) has a key role in the antivirus process, as it can increase the expression of antivirus-associated genes. Itaconate and its derivatives can regulate the immune response, secretion of inflammatory factors, and pyroptosis of macrophages. The effect of itaconate on IFN-β secretion of double-stranded RNA-induced macrophages are not well known. A derivative of itaconate, 4-octoyl itaconate (4-OI), was used to treat mouse bone marrow-derived macrophages (BMDM) induced with 100 μg/mL poly(I:C). The IFN-β concentration was detected through ELISA, and IFN-β mRNA expression was detected through quantitative PCR. High-throughput transcriptome sequencing was used to analyze changes in the BMDM transcriptome after 4-OI treatment. The Nrf2 expression was knocked down with siRNA.4-OI inhibited poly(I:C)-induced IFN-β secretion and mRNA expression in BMDM. Results of transcriptome sequencing revealed that 4-OI downregulated 1047 genes and upregulated 822 genes. GO and KEGG enrichment of differently expressed genes revealed that many downregulated genes were related to the anti-virus process, whereas many upregulated genes were related to metabolism. The Nrf2 inhibitor ML385 and Nrf2 siRNA could partially reverse the inhibitory effect of 4-OI. In conclusion, 4-octyl itaconate could inhibit the poly(I:C)-induced interferon-β secretion in BMDM partially by regulating Nrf2.

Abstract A070: Olaparib enhances radiation-induced Type I interferon and sensitizes pancreatic cancer to PD-L1 immune checkpoint inhibition

Abstract Inhibitors of PARP have been shown to sensitize pancreatic ductal adenocarcinoma (PDAC) to radiation therapy through mechanisms involving DNA damage, replication stress, and PARP trapping independent of BRCA1/2 mutation status. These mechanisms have the potential to activate innate immune signaling leading to Type I Interferon (T1IFN) production. Thus, we hypothesized that the clinical PARP1/2 inhibitor olaparib would potentiate radiation-induced T1IFN to activate an anti-tumoral immune response and sensitize resistant PDAC to immunotherapy. To test this hypothesis, we assessed the effects of olaparib and radiation on T1IFN production and signaling, tumor growth, sensitivity to PD-L1 immunotherapy, and the tumor immune microenvironment in mouse models of PDAC. We found that clinically achievable concentrations of olaparib significantly enhanced radiation-induced T1IFN production measured by both an IFNβ promoter-driven reporter and Ifnβ mRNA levels. Additionally, we found that the downstream effects of radiation-induced T1IFN signaling, including PD-L1 and MHC-I cell surface expression and induction of interferon stimulated genes including Cxcl9 and Cxcl10, were enhanced by olaparib. Co-treatment with a T1IFN receptor blocking antibody diminished these effects supporting the requirement for T1IFN-dependent signaling for the innate immune effects of radiation and olaparib. In vivo treatment with olaparib and radiation sensitized tumors to anti-PD-L1 therapy with a 40% complete response rate. These therapeutic responses led to an adaptive long term immunogenic memory evidenced by reduced engraftment following tumor rechallenge. Single-cell RNA sequencing revealed that treatment with olaparib, radiation and anti-PD-L1 augmented an anti-tumoral CD8+ T cell immune response, which is supported by an increase in CD8+ effector T cells and a decrease in CD8+ dysfunctional T cells. The necessity of functional CD8+ T cells to therapy efficacy in vivo was further reflected by a significantly reduced therapeutic combinational efficacy using a CD8+ T cell blocking antibody. Taken together, the results of this study show that the combination of olaparib, radiation and anti-PD-L1 induce an anti-tumoral immune response mediated by both T1IFN and CD8+ T cells. The results of this study provide the rationale for a phase 1 clinical trial (NCT05411094) combining dose escalated olaparib with durvalumab and radiotherapy in patients with locally advanced pancreatic cancer. Citation Format: Victoria M Valvo, Qiang Zhang, Long Jiang, Ashkay Tate, Michael D Green, Meredith A Morgan. Olaparib enhances radiation-induced Type I interferon and sensitizes pancreatic cancer to PD-L1 immune checkpoint inhibition [abstract]. In: Proceedings of the AACR-NCI-EORTC Virtual International Conference on Molecular Targets and Cancer Therapeutics; 2023 Oct 11-15; Boston, MA. Philadelphia (PA): AACR; Mol Cancer Ther 2023;22(12 Suppl):Abstract nr A070.

SARS-CoV-2 protein NSP2 enhances microRNA-mediated translational repression.

Viruses use microRNAs (miRNAs) to impair the host antiviral response and facilitate viral infection by expressing their own miRNAs or co-opting cellular miRNAs. miRNAs inhibit translation initiation of their target mRNAs by recruiting the GIGYF2/4EHP translation repressor complex to the mRNA 5´-cap structure. We recently reported that the severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) encoded non-structural protein 2 (NSP2) interacts with GIGYF2. This interaction is critical for blocking translation of the Ifn1-b mRNA that encodes the cytokine Interferon-ß, and thereby impairs the host antiviral response. However, it is not known whether NSP2 also affects miRNA-mediated silencing. Here, we demonstrate the pervasive augmentation of the miRNA-mediated translational repression of cellular mRNAs by NSP2. We show that NSP2 interacts with Argonaute 2, the core component of the miRNA-Induced Silencing Complex (miRISC) via GIGYF2 and enhances the translational repression mediated by natural miRNA binding sites in the 3´ UTR of cellular mRNAs. Our data reveal an additional layer of the complex mechanism by which SARS-CoV-2 and likely other coronaviruses manipulate the host gene expression program through co-opting the host miRNA-mediated silencing machinery.

Enzymolysis-based RNA pull-down identifies YTHDC2 as an inhibitor of antiviral innate response

The innate immune response must be terminated in a timely manner at the late stage of infection to prevent unwanted inflammation. The role of m6A-modified RNAs and their binding partners in this process is not well known. Here, we develop an enzymolysis-based RNA pull-down (eRP) method that utilizes the immunoglobulin G-degrading enzyme of Streptococcus pyogenes (IdeS) to fish out m6A-modified RNA-associated proteins. We apply eRP to capture the methylated single-stranded RNA (ssRNA) probe-associated proteins and identify YT521-B homology domain-containing 2 (YTHDC2) as the m6A-modified interferon β (IFN-β) mRNA-binding protein. YTHDC2, induced in macrophages at the late stage of virus infection, recruits IFN-stimulated exonuclease ISG20 (IFN-stimulated exonuclease gene 20) to degrade IFN-β mRNA, consequently inhibiting antiviral innate immune response. Invitro and invivo deficiency of YTHDC2 increases IFN-β production at the late stage of viral infection. Our findings establish an eRP method to effectively identify RNA-protein interactions and add mechanistic insight to the termination of innate response for maintaining homeostasis.

The D129L protein of African swine fever virus interferes with the binding of transcriptional coactivator p300 and IRF3 to prevent beta interferon induction.

African swine fever (ASF) is an acute, hemorrhagic, and severe porcine infectious disease caused by African swine fever virus (ASFV). ASF outbreaks severely threaten the global pig industries and result in serious economic losses. No safe and efficacious commercial vaccine is currently available except in Vietnam. To date, large gaps in the knowledge concerning viral biological characteristics and immunoevasion strategies have hindered the ASF vaccine design. In this study, we demonstrate that pD129L negatively regulates the type I interferon (IFN) signaling pathway by interfering with the interaction of the transcriptional coactivator p300 and IRF3, thereby inhibiting the induction of type I IFNs. This study reveals a novel immunoevasion strategy employed by ASFV, shedding new light on the intricate mechanisms for ASFV to evade the host immune responses.