Interferon Gene Expression Research Articles

Complement Suppresses the Initial Type 1 Interferon Response to Ocular Herpes Simplex Virus Type 1 Infection in Mice.

The complement system (CS) contributes to the initial containment of viral and bacterial pathogens and clearance of dying cells in circulation. We previously reported mice deficient in complement component 3 (C3KO mice) were more sensitive than wild-type (WT) mice to ocular HSV-1 infection, as measured by a reduction in cumulative survival and elevated viral titers in the nervous system but not the cornea between days three and seven post infection (pi). The present study was undertaken to determine if complement deficiency impacted virus replication and associated changes in inflammation at earlier time points in the cornea. C3KO mice were found to possess significantly (p < 0.05) less infectious virus in the cornea at 24 h pi that corresponded with a decrease in HSV-1 lytic gene expression at 12 and 24 h pi compared to WT animals. Flow cytometry acquisition found no differences in the myeloid cell populations residing in the cornea including total macrophage and neutrophil populations at 24 h pi with minimal infiltrating cell populations detected at the 12 h pi time point. Analysis of cytokine and chemokine content in the cornea measured at 12 and 24 h pi revealed that only CCL3 (MIP-1α) was found to be different between WT and C3KO mice with >2-fold increased levels (p < 0.05, ANOVA and Tukey's post hoc t-test) in the cornea of WT mice at 12 h pi. C3KO mouse resistance to HSV-1 infection at the early time points correlated with a significant increase in type I interferon (IFN) gene expression including IFN-α1 and IFN-β and downstream effector genes including tetherin and RNase L (p < 0.05, Mann-Whitney rank order test). These results suggest early activation of the CS interferes with the induction of the type I IFN response and leads to a transient increase in virus replication following corneal HSV-1 infection.

Cloning of human Type I interferon cDNAs.

In the late 1970s, crude interferon samples were found to exhibit anti-tumour activity. This discovery led to the interferon as a "magic drug" for cancer patients. Many groups, including those in Tokyo, Zürich, and San Francisco, attempted to identify human interferon cDNAs. Tadatsugu Taniguchi was the first to announce the cloning of human interferon-β cDNA in the December 1979 issue of Proc. Jpn. Acad. Ser. B. This was followed by the cloning of human interferon-α by a Zürich group and interferon-γ by a group in Genentech in San Francisco. Recombinant interferon proteins were produced on a large scale, and interferon-α was widely used to treat C-type hepatitis patients. The biological functions of interferons were quickly elucidated with the purified recombinant interferons. The molecular mechanisms underlying virus-induced interferon gene expression were also examined using cloned chromosomal genes. The background that led to interferon gene cloning and its impact on cytokine gene hunting is described herein.

Assessing the impact of dietary polystyrene nanoplastics on growth performance, immunological parameters, and antioxidant defense in zebrafish (Danio rerio)

AbstractThis trial was performed in order to investigate the response of zebrafish (Danio rerio) to dietary exposure to polystyrene nanoplastics (PS‐NPs) under laboratory conditions on fish growth and health. Healthy zebrafish (n=240) were divided into 12 tanks and fed with diets including 0 (T0), 100 (T1), 500 (T2), and 1000 (T3) mg kg−1 synthesized polystyrenes nanoplastics (PS‐NPs) for 30 days. At the end of trial, fish fed the PS‐NPs supplementation showed weight gain percentages of 79.45%, 70.35%, and 61.88% for T1, T2, and T3 groups, respectively, compared with 87.39% in the control fish. The SOD, GPX and MDA activities and cortisol levels increased by 47%, 32% and 35% and 46%, respectively, especially at high‐dose administration (p &lt; 0.05). The expression of GPX (T2‐81% and T3‐82%) and SOD (T2‐101% and T3‐187%) were remarkably upregulated in T2 and T3 groups. Moreover, the relative gene expression of HSP70, interleukin‐1 (IL1), Interferon γ (IFN‐γ) and tumor necrosis factor‐α (TNF‐α) increased by 178%, 202%, 154% and 307%, respectively, especially at high‐dose administration (p &lt; 0.05). The results of the present study demonstrated that exposure to PS‐NPs especially at high concentrations (500 and 1000 mg kg−1 of diet) negatively influenced growth, health status‐, antioxidant‐, and immunity‐related gene expression responses of zebrafish.

Weaning causes imbalanced T lymphocyte distribution and impaired intestinal immune barrier function in piglets

Context The immune system of piglets is not fully developed at weaning. The immature adaptive immune system along with the change in the feed types and the living conditions make piglets susceptible to pathogenic infections, and result in diarrhoea and decreased growth. However, the underlying mechanisms require further research. Aims This study was conducted to investigate the effects of weaning on the growth performance and intestinal immune function of piglets. Methods In total, 40 piglets with similar bodyweights were selected in pairs at 21 days old and divided into the suckling group (SG, breastfed by their mothers) and weaning group (WG, weaned at 21 days old). Eight piglets from each group were randomly selected and sacrificed at 24 days (SG3 and WG3) and 28 days of age (SG7 and WG7). The growth performance, T lymphocyte subpopulations, the concentration of cytokines and immunoglobulins, and the expression of Notch2 signalling proteins were determined. Key results The weaning caused a decrease in bodyweight (P &lt; 0.01) and the ratio of CD3+CD4+/CD3+CD8+ T cells in the thymus (P &lt; 0.05). Compared with SG3, the concentration of secretory immunoglobulin A (sIgA) in the jejunum was decreased, and that of interleukin 2 (IL-2) in serum and ileum, IL-1β and IL-2 in jejunum were upregulated (P &lt; 0.01), while IL-10 in the small intestine was downregulated (P &lt; 0.05) in WG3. Weaning downregulated gene expression of IL-4 and upregulated gene expression of IL-1β, IL-12, and interferon γ (IFN-γ) in small intestine (P &lt; 0.05). Weaning downregulated protein expression of Notch2 and Hes1 but upregulated Jagged1 expression in small intestine of piglets (P &lt; 0.05). Conclusions Weaning caused an imbalance in T lymphocyte distribution, thus impairing the intestinal immune function of piglets, which might be associated with the Notch2 signalling. Furthermore, weaning caused adverse effects on piglets especially on the first 3 days, which were partly recovered after 3 days, but still existed at 7 days after weaning. Implications The results of this study may guide the pig-raising industry, especially for the management of piglets shortly after weaning.

Shenqi Fuzheng Injection Reduces Cisplatin-Induced Kidney Injury via cGAS/STING Signaling Pathway in Breast Cancer Mice Model.

Shenqi Fuzheng Injection (SQFZ) is a traditional Chinese medicine injection consists of extracts of Codonopsis pilosula and Astragalus mongholicus. Combining SQFZ with conventional chemotherapy may improve the therapeutic efficacy and reduce side-effects of chemotherapy. However, the mechanisms of SQFZ reducing cisplatin-induced kidney injury are still unclear. The main compounds of SQFZ were identified via UPLC-Q-TOF-MS technique. Using multiple databases to predict potential targets for SQFZ. We established a breast cancer model by injecting 4T1 cells into mice. Tumor growth and body weight were observed. Serum blood urea nitrogen (BUN), creatinine (CRE), and glutathione (GSH) levels were measured. The extent of their kidney injury was measured by hematoxylin-eosin staining (HE). Cell apoptosis was identified using Hoechst33258 staining, flow cytometry and TUNEL. We evaluated H2AX and stimulator of interferon genes (STING) expression by immunohistochemistry (IHC), and assessed apoptosis-associated proteins by Western blotting analysis. We also evaluated mitochondrial function. The secretion of the inflammatory cytokines in serum was observed using ELISA assay. The effect of the STING pathway in HK-2 renal tubular epithelial cells exposed to cisplatin alone or combined with SQFZ. The potential targets of SQFZ on kidney injury mainly related to inflammatory responses, oxidation and antioxidant, apoptosis as well as IFN signaling pathway. Cisplatin significantly reduced animal weight, while there were no changes in the combination SQFZ and cisplatin. SQFZ counteracted cisplatin-induced BUN and CRE elevation. SQFZ ameliorated the oxidative stress induced by cisplatin. It diminished cisplatin-induced apoptosis and mitochondrial DNA damage and reversed cisplatin-induced cyclic guanosine monophosphate-adenosine monophosphate synthase (cGAS)/STING signaling pathway activation. It also improved the mitochondrial dysfunction induced by cisplatin. The results of the present study suggested that SQFZ effectively reduced cisplatin-induced kidney injury by inhibiting cGAS/STING signaling pathway.

Genome Wide siRNA Screen Identifies CGAS-Sting Pathway As a Pharmacological Target That Promotes Survival of Hematopoietic Stem Cells Deficient in Fanconi Genes

Introduction Fanconi anemia (FA) is a homozygous recessive genetic disorder due to mutation in the Fanconi family of genes. The disease is characterized by early onset bone marrow failure and a predisposition to cancer. The FA genes are involved in DNA damage sensing and repair. Mutation in these genes leads to heightened DNA damage and extreme sensitivity to cross-linking agents. Hematopoietic stem cells of FA patients are defective in differentiation and self-renewal, leading to stem cell exhaustion and bone marrow failure early in life. The current treatment for bone marrow failure is allogeneic stem cell transplant from healthy donors. Due to difficulties in finding matching donors and the inherent problems associated with allogeneic transplants, pharmacological therapies that delay or prevent bone marrow failure are of clinical importance. Other proposed therapies including TGF-b, metformin, and resveratrol have met with limited clinical success. Methodology: To identify genes that promote survival of FA cells we performed a genome wide siRNA screen using isogenic pair of cell line with FANCD2 deletions and its corrected counterpart using transfection of siRNA libraries targeting 18,119 genes from Human Drug Targets, Human Druggable Genome, and Human Genome libraries at 2000 cells/well in 384-well plates followed by cell viability measurement after 72 hrs. We measured IRF3 phosphorylation and expression of immune related genes to analyze the CGAS-STING pathway. Using peripheral blood and bone marrow derived CD34+ cells transduced with lentivirus expressing shRNA against FANCD2 we tested the effect of CGAS inhibitor in promoting CFU-GM and CFU-E. Results: A genome wide siRNA screen identified IRF7 as one of the top 5 hits whose diminished expression resulted in increased viability of F-D2 mutant PD20 cells. IRF7 along with IRF3 serve as downstream transcription factors including various Toll-like receptors, CGAS-STING pathways, and other pattern recognition innate immune receptors. Using pIRF3 as an activation readout for CGAS-STING pathway we identified that both FANCD2 and FANCA mutant cells have elevated pIRF3 levels. The elevated pIRF3 levels were further increased upon addition of mitomycin C (MMC) in FA mutant cells as compared to gene corrected cells. The pIRF3 levels decreased upon both genetic KD and small molecule inhibition of both CGAS and STING. In line with increased pIRF3 FA mutant cells showed increased gene expression of various interferon-regulated genes, which were then also reduced by CGAS inhibitor. Overexpression of RNAseH1 partially reduced pIRF3 levels in FA mutant cells indicating that the RNA:DNA hybrids whose levels are elevated in FA mutant cells is one of the ligands of CGAS. Most importantly 10µM of CGAS inhibitor RU.51 was able to rescue FA-A mutant cell line 6914 cells from low dose MMC-induced cell death showing that the sensitivity of FA mutant cells to DNA damaging agents was at least partially driven by CGAS-STING pathway. We then tested if the protective effect of CGAS inhibitor extended to human CD34+ cells deficient in FA genes. 20µM of RU.51 was able to rescue the CFU-GM and CFU-E defects of CD34+ cells carrying lentiviral integrated shRNA against FANCD2 to a level to that in cells expressing non-silencing shRNAs. Interferon-regulated gene expression was also elevated in the FANCD2-deficient CD34+ cells, which was reversed by treatment with cGAS inhibitor. Conclusions: It has previously been shown that FA patients have elevated TNF-alpha, IFN-gamma levels, and NFkbactivation, and interferon alpha administration hastens the onset of bone marrow failure in FA patients. Our results show that FA mutant cells have elevated CGAS-STING signaling resulting in heightened interferon gene expression. Elevated R-loop levels in FA cells act as trigger for the CGAS-STING pathway. CGAS inhibitor is able to rescue low dose MMC-induced cell death in FA mutant fibroblasts and also restores the CFU-GM and CFU-E forming ability of CD34+ cells. These results indicate that CGAS-STING pathway is a pharmacologically tractable target to reverse the hematopoietic stem cell exhaustion seen in FA patients.

Insulin resistance in macrophages impacts cholesterol biosynthesis and interferon gene expression and reduces foam cell formation capacity

Insulin resistance in macrophages impacts cholesterol biosynthesis and interferon gene expression and reduces foam cell formation capacity

Biodistribution of Fluorescent Albumin Nanoparticles among Organs of Laboratory Animals after Intranasal and Peroral Administration.

Natural, environmental and engineered nanoparticles (NP) penetrate into cells by endocytosis and induce innate immunity. The behaviour of the nanomaterials both in vitro and in vivo should be assessed. Our goal was to study protein NP stability in biological fluids and distribution in organs of animals after intranasal and oral administration. Bovine serum albumin (BSA) was labelled with the fluorescent dye RhoB and NP were fabricated by nanoprecipitation. The fluorescent protein NPwere administered intranasally and orally in laboratory-outbred mice ICR and rabbits. RhoB-BSA NP distribution in organs was detected using spectrofluorometry and fluorescent microscopy. Innate immunity was evaluated using reverse transcription with random hexanucleotide primer and subsequent real-time PCR with specific fluorescent hydrolysis probes. The labelled BSA NP were shown to remain stable in blood sera and nasopharyngeal swabs for 5 days at +37 °C. In vivo the maximal accumulation was found in the brain in 2 days posttreatment without prevalent accumulation in olfactory bulbs. For the intestine, heart and liver, the BSA NP accumulation was similar in 1 and 2 days, whereas for kidney samples even decreased after 1 day. Both intranasal and peroral administration of RhoB-BSA NP did not induce innate immunity. Thus, after intranasal or oral instillation RhoB-BSA NP were found mainly in the brain and intestine without interferon gene expression.

Phototherapy Restores Deficient Type I IFN Production and Enhances Antitumor Responses in Mycosis Fungoides

Transcriptional profiling demonstrated markedly reduced type I IFN gene expression in untreated mycosis fungoides (MF) skin lesions compared with that in healthy skin. Type I IFN expression in MF correlated with antigen-presenting cell-associated IRF5 before psoralen plus UVA therapy and epithelial ULBP2 after therapy, suggesting an enhancement of epithelial type I IFN. Immunostains confirmed reduced baseline type I IFN production in MF and increased levels after psoralen plus UVA treatment in responding patients. Effective tumor clearance was associated with increased type I IFN expression, enhanced recruitment of CD8+ T cells into skin lesions, and expression of genes associated with antigen-specific T-cell activation. IFNk, a keratinocyte-derived inducer of type I IFNs, was increased by psoralen plus UVA therapy and expression correlated with upregulation of other type I IFNs. Invitro, deletion of keratinocyte IFNk decreased baseline and UVA-induced expression of type I IFN and IFN response genes. In summary, we find a baseline deficit in type I IFN production in MF that is restored by psoralen plus UVA therapy and correlates with enhanced antitumor responses. This may explain why MF generally develops in sun-protected skin and suggests that drugs that increase epithelial type I IFNs, including topical MEK and EGFR inhibitors, may be effective therapies for MF.

IL-6 trans-signaling in a humanized mouse model of scleroderma

Fibrosis is regulated by interactions between immune and mesenchymal cells. However, the capacity of cell types to modulate human fibrosis pathology is poorly understood due to lack of a fully humanized model system. MISTRG6 mice were engineered by homologous mouse/human gene replacement to develop an immune system like humans when engrafted with human hematopoietic stem cells (HSCs). We utilized MISTRG6 mice to model scleroderma by transplantation of healthy or scleroderma skin from a patient with pansclerotic morphea to humanized mice engrafted with unmatched allogeneic HSC. We identified that scleroderma skin grafts contained both skin and bone marrow-derived human CD4 and CD8 T cells along with human endothelial cells and pericytes. Unlike healthy skin, fibroblasts in scleroderma skin were depleted and replaced by mouse fibroblasts. Furthermore, HSC engraftment alleviated multiple signatures of fibrosis, including expression of collagen and interferon genes, and proliferation and activation of human T cells. Fibrosis improvement correlated with reduced markers of T cell activation and expression of human IL-6 by mesenchymal cells. Mechanistic studies supported a model whereby IL-6 trans-signaling driven by CD4 T cell-derived soluble IL-6 receptor complexed with fibroblast-derived IL-6 promoted excess extracellular matrix gene expression. Thus, MISTRG6 mice transplanted with scleroderma skin demonstrated multiple fibrotic responses centered around human IL-6 signaling, which was improved by the presence of healthy bone marrow-derived immune cells. Our results highlight the importance of IL-6 trans-signaling in pathogenesis of scleroderma and the ability of healthy bone marrow-derived immune cells to mitigate disease.

SARS-CoV-2 Mac1 is required for IFN antagonism and efficient virus replication in cell culture and in mice

Several coronavirus (CoV) encoded proteins are being evaluated as targets for antiviral therapies for COVID-19. Included in these drug targets is the conserved macrodomain, or Mac1, an ADP-ribosylhydrolase and ADP-ribose binding protein encoded as a small domain at the N terminus of nonstructural protein 3. Utilizing point mutant recombinant viruses, Mac1 was shown to be critical for both murine hepatitis virus (MHV) and severe acute respiratory syndrome (SARS)-CoV virulence. However, as a potential drug target, it is imperative to understand how a complete Mac1 deletion impacts the replication and pathogenesis of different CoVs. To this end, we created recombinant bacterial artificial chromosomes (BACs) containing complete Mac1 deletions (ΔMac1) in MHV, MERS-CoV, and SARS-CoV-2. While we were unable to recover infectious virus from MHV or MERS-CoV ΔMac1 BACs, SARS-CoV-2 ΔMac1 was readily recovered from BAC transfection, indicating a stark difference in the requirement for Mac1 between different CoVs. Furthermore, SARS-CoV-2 ΔMac1 replicated at or near wild-type levels in multiple cell lines susceptible to infection. However, in a mouse model of severe infection, ΔMac1 was quickly cleared causing minimal pathology without any morbidity. ΔMac1 SARS-CoV-2 induced increased levels of interferon (IFN) and IFN-stimulated gene expression in cell culture and mice, indicating that Mac1 blocks IFN responses which may contribute to its attenuation. ΔMac1 infection also led to a stark reduction in inflammatory monocytes and neutrophils. These results demonstrate that Mac1 only minimally impacts SARS-CoV-2 replication, unlike MHV and MERS-CoV, but is required for SARS-CoV-2 pathogenesis and is a unique antiviral drug target.

Interferon-stimulated gene 15 facilitates BTV replication through interacting with the NS1 protein.

Bluetongue virus (BTV) infection effectively activates the innate immune response, followed by the expression of interferon (IFN) and multiple interferon-stimulated genes (ISGs). ISG15 is one of the most induced ISGs, and often plays a role in inhibiting virus replication. This study aims to explore the role and specific mechanisms of ovine ISG15 (oISG15) in BTV infection. We found that the transcription level of oISG15 was upregulated in a time-dependent and BTV multiplicity of infection-dependent manner. The overexpression of exogenous oISG15 enhances BTV replication, whereas the knockdown of endogenous oISG15 inhibits BTV replication. The viral protein in wild-type oISG15-overexpressed cells and ISGylation defective oISG15-overexpressed cells have no significant differences, which indicated that oISG15 promoted BTV replication in an ISGylation-independent manner. A co-immunoprecipitation assay showed that four viral BTV proteins-VP3, VP4, VP5, and NS1-interacted with oISG15. We also found that the VP4 and NS1 proteins associated with ubiquitin via co-immunoprecipitation, and that oISG15 overexpression improved the stability of both proteins. Further results showed that the degradation of NS1 was involved in lysine 63-linked polyubiquitin. This suggested that oISG15 may interfere with NS1 degradation via the autophagy pathway. This study provides new insights on the interaction between BTV and ISG15, and enriches our understanding of the regulation and biological function of ISG15 with virus replication.

Interferonopathies masquerading as non-Mendelian autoimmune diseases: pattern recognition for early diagnosis.

Type I interferonopathies are a broad category of conditions associated with increased type I interferon gene expression and include monogenic autoinflammatory diseases and non-Mendelian autoimmune diseases such as dermatomyositis and systemic lupus erythematosus. While a wide range of clinical presentations among type I interferonopathies exists, these conditions often share several clinical manifestations and implications for treatment. Presenting symptoms may mimic non-Mendelian autoimmune diseases, including vasculitis and systemic lupus erythematosus, leading to delayed or missed diagnosis. This review aims to raise awareness about the varied presentations of monogenic interferonopathies to provide early recognition and appropriate treatment to prevent irreversible damage and improve quality of life and outcomes in this unique patient population.

RSV replication modifies the XBP1s binding complex on the IRF1 upstream enhancer to potentiate the mucosal anti-viral response.

The unfolded protein response (UPR) has emerged as an important signaling pathway mediating anti-viral defenses to Respiratory Syncytial Virus (RSV) infection. Earlier we found that RSV replication predominantly activates the evolutionarily conserved Inositol Requiring Enzyme 1α (IRE1α)-X-Box Binding Protein 1 spliced (XBP1s) arm of the Unfolded Protein Response (UPR) producing inflammation, metabolic adaptation and cellular plasticity, yet the mechanisms how the UPR potentiates inflammation are not well understood. To understand this process better, we examined the genomic response integrating RNA-seq and Cleavage Under Targets and Release Using Nuclease (CUT&RUN) analyses. These data were integrated with an RNA-seq analysis conducted on RSV-infected small airway cells ± an IRE1α RNAse inhibitor. We identified RSV induced expression changes in ~3.2K genes; of these, 279 required IRE1α and were enriched in IL-10/cytokine signaling pathways. From this data set, we identify those genes directly under XBP1s control by CUT&RUN. Although XBP1s binds to ~4.2 K high-confidence genomic binding sites, surprisingly only a small subset of IL10/cytokine signaling genes are directly bound. We further apply CUT&RUN to find that RSV infection enhances XBP1s loading on 786 genomic sites enriched in AP1/Fra-1, RELA and SP1 binding sites. These control a subset of cytokine regulatory factor genes including IFN response factor 1 (IRF1), CSF2, NFKB1A and DUSP10. Focusing on the downstream role of IRF1, selective knockdown (KD) and overexpression experiments demonstrate IRF1 induction controls type I and -III interferon (IFN) and IFN-stimulated gene (ISG) expression, demonstrating that ISG are indirectly regulated by XBP1 through IRF1 transactivation. Examining the mechanism of IRF1 activation, we observe that XBP1s directly binds a 5' enhancer sequence whose XBP1s loading is increased by RSV. The functional requirement for the enhancer is demonstrated by targeting a dCas9-KRAB silencer, reducing IRF1 activation. Chromatin immunoprecipitation shows that XBP1 is required, but not sufficient, for RSV-induced recruitment of activated phospho-Ser2 Pol II to the enhancer. We conclude that XBP1s is a direct activator of a core subset of IFN and cytokine regulatory genes in response to RSV. Of these IRF1 is upstream of the type III IFN and ISG response. We find that RSV modulates the XBP1s binding complex on the IRF1 5' enhancer whose activation is required for IRF1 expression. These findings provide novel insight into how the IRE1α-XBP1s pathway potentiates airway mucosal anti-viral responses.

The Cytomegalovirus M35 Protein Directly Binds to the Interferon-β Enhancer and Modulates Transcription of Ifnb1 and Other IRF3-Driven Genes.

Induction of type I interferon (IFN) gene expression is among the first lines of cellular defense a virus encounters during primary infection. We previously identified the tegument protein M35 of murine cytomegalovirus (MCMV) as an essential antagonist of this antiviral system, showing that M35 interferes with type I IFN induction downstream of pattern-recognition receptor (PRR) activation. Here, we report structural and mechanistic details of M35's function. Determination of M35's crystal structure combined with reverse genetics revealed that homodimerization is a key feature for M35's immunomodulatory activity. In electrophoretic mobility shift assays (EMSAs), purified M35 protein specifically bound to the regulatory DNA element that governs transcription of the first type I IFN gene induced in nonimmune cells, Ifnb1. DNA-binding sites of M35 overlapped with the recognition elements of interferon regulatory factor 3 (IRF3), a key transcription factor activated by PRR signaling. Chromatin immunoprecipitation (ChIP) showed reduced binding of IRF3 to the host Ifnb1 promoter in the presence of M35. We furthermore defined the IRF3-dependent and the type I IFN signaling-responsive genes in murine fibroblasts by RNA sequencing of metabolically labeled transcripts (SLAM-seq) and assessed M35's global effect on gene expression. Stable expression of M35 broadly influenced the transcriptome in untreated cells and specifically downregulated basal expression of IRF3-dependent genes. During MCMV infection, M35 impaired expression of IRF3-responsive genes aside of Ifnb1. Our results suggest that M35-DNA binding directly antagonizes gene induction mediated by IRF3 and impairs the antiviral response more broadly than formerly recognized. IMPORTANCE Replication of the ubiquitous human cytomegalovirus (HCMV) in healthy individuals mostly goes unnoticed but can impair fetal development or cause life-threatening symptoms in immunosuppressed or -deficient patients. Like other herpesviruses, CMV extensively manipulates its hosts and establishes lifelong latent infections. Murine CMV (MCMV) presents an important model system as it allows the study of CMV infection in the host organism. We previously showed that during entry into host cells, MCMV virions release the evolutionary conserved protein M35 protein to immediately dampen the antiviral type I interferon (IFN) response induced by pathogen detection. Here, we show that M35 dimers bind to regulatory DNA elements and interfere with recruitment of interferon regulatory factor 3 (IRF3), a key cellular factor for antiviral gene expression. Thereby, M35 interferes with expression of type I IFNs and other IRF3-dependent genes, reflecting the importance for herpesviruses to avoid IRF3-mediated gene induction.

Anthracyclines inhibit SARS-CoV-2 infection

Vaccines and drugs are two effective medical interventions to mitigate SARS-CoV-2 infection. Three SARS-CoV-2 inhibitors, remdesivir, paxlovid, and molnupiravir, have been approved for treating COVID-19 patients, but more are needed, because each drug has its limitation of usage and SARS-CoV-2 constantly develops drug resistance mutations. In addition, SARS-CoV-2 drugs have the potential to be repurposed to inhibit new human coronaviruses, thus help to prepare for future coronavirus outbreaks. We have screened a library of microbial metabolites to discover new SARS-CoV-2 inhibitors. To facilitate this screening effort, we generated a recombinant SARS-CoV-2 Delta variant carrying the nano luciferase as a reporter for measuring viral infection. Six compounds were found to inhibit SARS-CoV-2 at the half maximal inhibitory concentration (IC50) below 1 μM, including the anthracycline drug aclarubicin that markedly reduced viral RNA-dependent RNA polymerase (RdRp)-mediated gene expression, whereas other anthracyclines inhibited SARS-CoV-2 by activating the expression of interferon and antiviral genes. As the most commonly prescribed anti-cancer drugs, anthracyclines hold the promise of becoming new SARS-CoV-2 inhibitors.

Reduced NR2F2 Expression in the Host Response to Infectious Bursal Disease Virus Infection Suppressed Viral Replication by Enhancing Type I Interferon Expression by Targeting SOCS5.

Nuclear receptors are ligand-activated transcription factors that play an important role in regulating innate antiviral immunity and other biological processes. However, the role of nuclear receptors in the host response to infectious bursal disease virus (IBDV) infection remains elusive. In this study, we show that IBDV infection or poly(I·C) treatment of DF-1 or HD11 cells markedly decreased nuclear receptor subfamily 2 group F member 2 (NR2F2) expression. Surprisingly, knockdown, knockout, or inhibition of NR2F2 expression in host cells remarkably inhibited IBDV replication and promoted IBDV/poly(I·C)-induced type I interferon and interferon-stimulated genes expression. Furthermore, our data show that NR2F2 negatively regulates the antiviral innate immune response by promoting the suppressor of cytokine signaling 5 (SOCS5) expression. Thus, reduced NR2F2 expression in the host response to IBDV infection inhibited viral replication by enhancing the expression of type I interferon by targeting SOCS5. These findings reveal that NR2F2 plays a crucial role in antiviral innate immunity, furthering our understanding of the mechanism underlying the host response to viral infection. IMPORTANCE Infectious bursal disease (IBD) is an immunosuppressive disease causing considerable economic losses to the poultry industry worldwide. Nuclear receptors play an important role in regulating innate antiviral immunity. However, the role of nuclear receptors in the host response to IBD virus (IBDV) infection remains elusive. Here, we report that NR2F2 expression decreased in IBDV-infected cells, which consequently reduced SOCS5 expression, promoted type I interferon expression, and suppressed IBDV infection. Thus, NR2F2 serves as a negative factor in the host response to IBDV infection by regulating SOCS5 expression, and intervention in the NR2F2-mediated host response by specific inhibitors might be employed as a strategy for prevention and treatment of IBD.

Gene expression of pregnancy-associated glycoproteins-1 (PAG-1), interferon-tau (IFNt) and interferon stimulated genes (ISGs) as diagnostic and prognostic markers of maternal-fetal cellular interaction in buffalo cows

The aim of this study was to determine the presence of Pregnancy-associated glycoprotein −1 (PAG-1) mRNA expression in the maternal circulation of pregnant buffaloes during the early stage of pregnancy. Contemporaneously, the mRNA expression levels of Interferon-tau (IFNt) and some Interferon stimulated genes (ISGs) (interferon stimulated gene 15 ubiquitin-like modifier interferon, ISG15; Mixoviruses resistance 1 and 2, MX1 and MX2; 2′,5′-oligoadenylate synthase 1,OAS1) were evaluated in order to expand our knowledge of the molecular processes involved in the early stages of pregnancy and to identify potential biomarkers of maternal-fetal cellular interaction in buffalo. The study was conducted on 38 synchronized and artificially inseminated buffalo cows (d 0), divided ex post into 3 groups: Pregnant (n = 17), Non-pregnant (n = 15) and Embryo mortality (n = 6). Blood samples were collected on d 14, 19, 28 and 40 after artificial insemination (AI) for peripheral blood mononuclear cells (PBMCs) isolation. Expression levels of mRNA of PAG-1, IFNt, ISG15. MX1, MX2 and OAS1 were measured using RT-qPCR. No significant changes were observed in IFNt and PAG gene expressions between groups, while significant differences (p < 0.001) were found for ISG15, MX1, MX2, and OAS1. Pairwise comparisons revealed that the differences between groups occurred on days 19 and 28 post-AI. ISG15 proved to have the best diagnostic performance for distinguishing between pregnant animals and animals that experienced embryo mortality with the ROC analysis. According to the results of the univariate analyses, day 19 was identified as the most indicative to discriminate between groups while the most reliable genes for this differentiation were ISG15, MX1 and MX2. MX2 proved to be the best gene for discriminating pregnant buffaloes using the discriminant analysis, while MX1 was the gene that best predicted embryo mortality. Our results showed that among PAG-1, IFNt and ISGs expression as diagnostic and prognostic markers of maternal-fetal cellular interaction in buffalo cows, ISGs proved to be the best peripheral biomarkers for predicting pregnancy and embryonic mortality during the peri-implantation period. These insights into the mechanisms behind maternal-fetal interaction and the development of a method for the early detection of embryo distress may enable us to implement effective strategies to support embryo survival.

Stimulator of interferon gene (STING) expression as a biomarker for overall survival in PDL1-negative, TMB-low non-small cell lung cancer (NSCLC) treated with immune checkpoint inhibitors (ICIs).

9120 Background: cGAS-STING (CGS) is an intracellular DNA sensing pathway that when activated, stimulates interferon and other immune-related signaling pathways. While increased expression of CGS-related genes has been associated with better survival in NSCLC, studies investigating this phenomenon in tumors less likely to respond to ICIs such as PDL1-negative, TMB-Low NSCLC are lacking. Here we report the role of CGS pathway in NSCLC tumors that are both PDL1-negative and TMB-Low. Methods: DNA (592-gene or whole exome) and RNA sequencing (whole transcriptome) was performed for NSCLC specimens (n=7264) submitted to Caris Life Sciences (Phoenix, AZ). PDL1 negativity was defined as &lt;1% TPS score (22C3) &amp; TMB-Low as &lt;10 mutations/Mb. Hierarchical Agglomerative Clustering (HAC) was performed on STING pathway genes (CXCL10, CCL5 and GZB) and tumors were grouped into STING-high (n=3070), STING-intermediate (n=1609) and STING-low (n=2585) groups. Microenvironmental cell populations (MCP)-counter analysis was used to estimate cellular composition of immune cells. Statistical significance was determined using chi-square/Fisher’s exact/Mann-Whitney U tests and adjusted for multiple comparisons (q&lt;0.05). Kaplan Meier method was used to estimate the overall survival (OS) derived from insurance claims of IO treated pts with NSCLC (n=400). Pts were stratified into groups above or below the median expression of the STING gene ( TMEM173). Results: Compared to STING-low, tumors that were STING-high had enrichment in immune cell infiltrates including CD8+ T cells (4.4-fold) and B-cells (5.9-fold); Additionally, STING-high cohort had significantly higher expression of immune checkpoint (IC) genes including CD274(2.5-fold), CD80 (2.7-fold, all q&lt;0.05). STING-high tumors also had a higher prevalence of TP53 mutations (mts) and a lower prevalence of STK11 and KEAP1 mts (Table). Median overall survival (OS) was 21.1 months in the STING-high cohort (n=200) vs 11.9 months in the STING-low cohort (n=199) in IO treated pts (HR = 0.62, p&lt;0.000.1) Conclusions: To our knowledge this is the largest real-world dataset indicating that enrichment of both the STING gene, and gene signatures associated with the STING pathway represent valuable transcriptomic biomarkers to stratify pts with PDL1-negative and TMB-Low NSCLC who benefit more from IO therapies. Conversely, novel IO combination strategies need to be employed in STING-low tumors to enhance outcomes. [Table: see text]

Context-dependent activation of STING-interferon signaling by CD11b agonists enhances anti-tumor immunity

Chronic activation of inflammatory pathways and suppressed interferon are hallmarks of immunosuppressive tumors. Previous studies have shown that CD11b integrin agonists could enhance anti-tumor immunity through myeloid reprograming, but the underlying mechanisms remain unclear. Herein we find that CD11b agonists alter tumor-associated macrophage (TAM) phenotypes by repressing NF-κB signaling and activating interferon gene expression simultaneously. Repression of NF-κB signaling involves degradation of p65 protein and is context independent. In contrast, CD11b agonism induces STING/STAT1 pathway-mediated interferon gene expression through FAK-mediated mitochondrial dysfunction, with the magnitude of induction dependent on the tumor microenvironment and amplified by cytotoxic therapies. Using tissues from phase I clinical studies, we demonstrate that GB1275 treatment activates STING and STAT1 signaling in TAMs in human tumors. These findings suggest potential mechanism-based therapeutic strategies for CD11b agonists and identify patient populations more likely to benefit.