Expression Of Retinoic Acid-inducible gene-I Research Articles

Involvement of retinoic acid‑inducible gene‑I in radiation‑induced senescence of human umbilical vein endothelial cells.

In 2012, the threshold radiation dose (0.5 Gy) for cardiovascular and cerebrovascular diseases was revised, and this threshold dose may be exceeded during procedures involving radiation such as interventional radiology. Therefore, in addition to regulating radiation dose, it is necessary to develop strategies to prevent and mitigate the development of cardiovascular disease. Cellular senescence is irreversible arrest of cell proliferation. Although cellular senescence is one of the mechanisms for suppressing cancer, it also has adverse effects. For example, senescence of vascular endothelial cells is involved in development of vascular disorders. However, the mechanisms underlying induction of cellular senescence are not fully understood. Therefore, the present study explored the factors involved in the radiation-induced senescence in human umbilical vein endothelial cells (HUVECs). The present study reanalyzed the gene expression data of senescent normal human endothelial cells and fibroblast after irradiation (NCBI Gene Expression Omnibus accession no. GSE130727) and microarray data of HUVECs 24 h after irradiation (NCBI Gene Expression Omnibus accession no. GSE76484). Numerous genes related to viral infection and inflammation were upregulated in radiation-induced senescent cells. In addition, the gene group involved in the retinoic acid-inducible gene-I (RIG-I)-like receptor (RLR) signaling pathway, which plays an important role to induce anti-viral response, was altered in irradiated HUVECs. Therefore, to investigate the involvement of RIG-I and melanoma differentiation-associated gene 5 (MDA5), which are RLRs, in radiation-induced senescence of HUVECs, the protein expression of RIG-I and MDA5 and the activity of senescence-associated β-galactosidase (SA-β-gal), a representative senescence marker, were analyzed. Of note, knockdown of RIG-I in HUVECs significantly decreased radiation-increased proportion of cells with high SA-β-gal activity (i.e., senescent cells), whereas this phenomenon was not observed in MDA5-knockdown cells. Taken together, the present results suggested that RIG-I, but not MDA5, was associated with radiation-induced senescence in HUVECs.

RIG-I promotes cell proliferation in esophageal squamous cell carcinoma by facilitating p21 degradation.

Retinoic acid-inducible gene-I (RIG-I) is considered a key sensor for host recognition of RNA virus infections. Recent studies have shown that RIG-I also regulates carcinogenesis. However, the role of RIG-I in esophageal squamous cell carcinoma (ESCC) remains unclear. We investigated the RIG-I expression in ESCC cells using a public database, immunohistochemistry, and Western blotting. We evaluated the proliferative activity of ESCC cells using CCK-8, colony formation, and EdU staining assays. Further, we determined the ESCC cell-cycle changes using flow cytometry and the ubiquitination of p21 in the cells using cycloheximide chase and ubiquitination assays. Finally, we verified the in vivo effects of RIG-I on ESCC cells by constructing xenograft models. RIG-I was highly expressed in ESCC cells and significantly promoted their proliferation and cell-cycle. Moreover, RIG-I knockdown inhibited xenograft growth in nude mice. Furthermore, RIG-I accelerated the cell-cycle by promoting the ubiquitination and degradation of p21. Overall, this study revealed that the increased expression of RIG-I due to ESCC accelerated the progression of esophageal cancer by promoting the ubiquitination and degradation of p21, which is related to the prognosis of ESCC. Thus, RIG-I may be a novel therapeutic target for ESCC treatment.

RIG-I Promotes Tumorigenesis and Confers Radioresistance of Esophageal Squamous Cell Carcinoma by Regulating DUSP6.

We investigated the expression and biological function of retinoic acid inducible gene I (RIG-I) in esophageal squamous cell carcinoma (ESCC). Materials and methods: An immunohistochemical analysis was performed on 86 pairs of tumor tissue and adjacent normal tissue samples of patients with ESCC. We generated RIG-I-overexpressing ESCC cell lines KYSE70 and KYSE450, and RIG-I- knockdown cell lines KYSE150 and KYSE510. Cell viability, migration and invasion, radioresistance, DNA damage, and cell cycle were evaluated using CCK-8, wound-healing and transwell assay, colony formation, immunofluorescence, and flow cytometry and Western blotting, respectively. RNA sequencing was performed to determine the differential gene expression between controls and RIG-I knockdown. Tumor growth and radioresistance were assessed in nude mice using xenograft models. RIG-I expression was higher in ESCC tissues compared with that in matched non-tumor tissues. RIG-I overexpressing cells had a higher proliferation rate than RIG-I knockdown cells. Moreover, the knockdown of RIG-I slowed migration and invasion rates, whereas the overexpression of RIG-I accelerated migration and invasion rates. RIG-I overexpression induced radioresistance and G2/M phase arrest and reduced DNA damage after exposure to ionizing radiations compared with controls; however, it silenced the RIG-I enhanced radiosensitivity and DNA damage, and reduced the G2/M phase arrest. RNA sequencing revealed that the downstream genes DUSP6 and RIG-I had the same biological function; silencing DUSP6 can reduce the radioresistance caused by the overexpression of RIG-I. RIG-I knockdown depleted tumor growth in vivo, and radiation exposure effectively delayed the growth of xenograft tumors compared with the control group. RIG-I enhances the progression and radioresistance of ESCC; therefore, it may be a new potential target for ESCC-targeted therapy.

Immunoproteasome subunit β5i promotes perifascicular muscle atrophy in dermatomyositis by upregulating RIG-I

BackgroundPerifascicular atrophy is a unique pathological hallmark in dermatomyositis (DM)-affected muscles; however, the mechanism underlying this process remains unclear. In this study, we aimed to investigate the potential role of...

JMJD4-demethylated RIG-I prevents hepatic steatosis and carcinogenesis

BackgroundHepatocarcinogenesis is driven by necroinflammation or metabolic disorders, and the underlying mechanisms remain largely elusive. We previously found that retinoic acid-inducible gene-I (RIG-I), a sensor for recognizing RNA virus in innate immune cells, is mainly expressed by parenchymal hepatocytes in the liver. However, its roles in hepatocarcinogenesis are unknown, which is intensively investigated in this study.MethodsDEN-induced necroinflammation-driven hepatocarcinogenesis and STAM NASH-hepatocarcinogenesis were carried out in hepatocyte-specific RIG-I knockout mice. The post-translational modification of RIG-I was determined by mass spectrometry, and specific antibodies against methylated lysine sites and the RIG-I lysine mutant mice were constructed to identify the functions of RIG-I methylation.ResultsWe interestingly found that DEN-induced hepatocarcinogenesis was enhanced, while NASH-induced hepatocarcinogenesis was suppressed by hepatocyte-specific RIG-I deficiency. Further, IL-6 decreased RIG-I expression in HCC progenitor cells (HcPCs), which then viciously promoted IL-6 effector signaling and drove HcPCs to fully established HCC. RIG-I expression was increased by HFD, which then enhanced cholesterol synthesis and steatosis, and the in-turn NASH and NASH-induced hepatocarcinogenesis. Mechanistically, RIG-I was constitutively mono-methylated at K18 and K146, and demethylase JMJD4-mediated RIG-I demethylation suppressed IL-6-STAT3 signaling. The constitutive methylated RIG-I associated with AMPKα to inhibit HMGCR phosphorylation, thus promoting HMGCR enzymatic activity and cholesterol synthesis. Clinically, RIG-I was decreased in human hepatic precancerous dysplastic nodules while increased in NAFLD livers, which were in accordance with the data in mouse models.ConclusionsDecreased RIG-I in HcPCs promotes necroinflammation-induced hepatocarcinogenesis, while increased constitutive methylated RIG-I enhances steatosis and NASH-induced hepatocarcinogenesis. JMJD4-demethylated RIG-I prevents both necroinflammation and NASH-induced hepatocarcinogenesis, which provides mechanistic insight and potential target for preventing HCC.

Transcriptome Analysis of Retinoic Acid-Inducible Gene I Overexpression Reveals the Potential Genes for Autophagy-Related Negative Regulation.

Retinoic acid-inducible gene I (RIG-I) serves as an essential viral RNA sensor for innate immune. The activation of the RIG-I-like receptors (RLRs) pathway triggers many regulations for the outcome of type I interferon, including ubiquitination, dephosphorylation, ISGylation, and autophagy. However, the autophagy-related regulation of RIG-I is still not fully understood. To investigate the potentially unknown genes related to autophagy-related regulation of RIG-I, we firstly confirm the induction of autophagy derived by overexpression of RIG-I. Furthermore, the autophagy inducer and inhibitor drugs were used in different assays. The results showed autophagy could control the activation of RLRs pathway and expression of exogenous RIG-I. In addition, we carried out the transcriptome analysis of overexpression of RIG-I in vitro. Differentially expressed genes (DEGs) in GO and KEGG signaling pathways enrichment provided a newly complex network. Finally, the validation of qPCR indicated that the DEGs PTPN22, PRKN, OTUD7B, and SIRT2 were correlated to the negative regulation of excessive expression of RIG-I. Taken together, our study contributed new insights into a more comprehensive understanding of the regulation of excessive expression of RIG-I. It provided the potential candidate genes for autophagy-related negative regulation for further investigation.

QKI deficiency in macrophages protects mice against JEV infection by regulating cell migration and antiviral response

Japanese encephalitis (JE) is a major reason to cause viral encephalitis, with 50% patients suffering from severe neuro-inflammation and permanent neural injury. Effective anti-viral treatment is urgently needed. Here, we found RNA binding protein quaking (QKI) was involved in the progression of JE by regulating migration and anti-viral response of macrophages. After JE virus (JEV) infection, QKI-deficient mice had lower viral loads in the brain and fewer neurological symptoms. In comparison with control mice, proinflammatory cytokines in the brain of QKI-deficient animals revealed distinct patterns, with lower levels of IL-6 (interleukin-6) and IFN-β (interferon-β) at the early stage but higher levels at the end of JE. Then we found infiltration of CCR2 positive ((C-C motif) receptor 2) peripheral macrophages and CCR2 expression on macrophages were inhibited in QKI-deficient mice, while the expression of CCR2 ligands was not changed. Bioinformatical analysis showed that a QRE (quaking response element) located on 3′UTR (untranslated region) of Ccr2. We further verified that QKI was able to interact with Ccr2 mRNA and regulate its degradation in vitro. Additionally, since the IFN-β production was increased in QKI-ablation mice after JEV infection, the anti-viral response was analyzed. Results in QKI-silenced N9 cells showed that the expression of RIG-I (retinoic acid-inducible gene-I) and TBK1 (TANK binding kinase 1) was increased, thus further inducing IRF3 (interferon regulatory factor 3) phosphorylation and interferon activation. Overall, these results revealed QKI mediated the anti-viral process via interfering migration of macrophages to CNS (central nervous system) and enhancing RIG-I/IRF3/IFN-β pathway to restrict virus dissemination.

The intrinsically disordered CARDs-Helicase linker in RIG-I is a molecular gate for RNA proofreading.

The innate immune receptor RIG‐I provides a first line of defense against viral infections. Viral RNAs are recognized by RIG‐I's C‐terminal domain (CTD), but the RNA must engage the helicase domain to release the signaling CARD (Caspase Activation and Recruitment Domain) domains from their autoinhibitory CARD2:Hel2i interactions. Because the helicase itself lacks RNA specificity, mechanisms to proofread RNAs entering the helicase domain must exist. Although such mechanisms would be crucial in preventing aberrant immune responses by non‐specific RNAs, they remain largely uncharacterized to date. This study reveals a previously unknown proofreading mechanism through which RIG‐I ensures that the helicase engages RNAs explicitly recognized by the CTD. A crucial part of this mechanism involves the intrinsically disordered CARDs‐Helicase Linker (CHL), which connects the CARDs to the helicase subdomain Hel1. CHL uses its negatively charged regions to antagonize incoming RNAs electrostatically. In addition to this RNA gating function, CHL is essential for stabilization of the CARD2:Hel2i interface. Overall, we uncover that the CHL and CARD2:Hel2i interface work together to establish a tunable gating mechanism that allows CTD‐chosen RNAs to bind the helicase domain, while at the same time blocking non‐specific RNAs. These findings also indicate that CHL could represent a novel target for RIG‐I‐based therapeutics.

The RIG-I–NRF2 axis regulates the mesenchymal stromal niche for bone marrow transplantation

AbstractBone marrow–derived mesenchymal stem cells (BMSCs) support bone formation and constitute the stromal niche in regulating hematopoietic stem cells (HSCs). Stromal niche dysfunction affects HSC engraftment during transplantation; however, the underlying mechanisms remain elusive. In the present study, we found that all-trans retinoic acid (ATRA) and inflammation stress upregulated retinoic acid–inducible gene I (RIG-I) in BMSCs. Excess RIG-I expression damaged the clonogenicity, bone-forming ability of BMSCs and particularly their stromal niche function that supports HSC expansion in vitro and engraftment in vivo. Mechanistically, RIG-I elevation promoted the degradation of NRF2, a checkpoint for antioxidant cellular response, by altering the RIG-I-Trim25-Keap1-NRF2 complex, leading to reactive oxygen species (ROS) accumulation and BMSC damage. Genetic inhibition of RIG-I sustained NRF2 protein levels and reduced ROS levels in ATRA-treated BMSCs, thus preserving their clonogenicity, bone-forming ability, and stromal niche function in supporting HSC engraftment in mice. More importantly, RIG-I inhibition recovered the ATRA-treated stromal niche function to enhance HSC engraftment and emergency myelopoiesis for innate immunity against the bacterium Listeria monocytogenes during transplantation. Overall, we identified a noncanonical role of RIG-I in the regulation of the stromal niche for HSC transplantation.

The Variation of Duck RIG-I-Mediated Innate Immune Response Induced by Different Virulence Avian Influenza Viruses.

In recent years, the emerging highly pathogenic avian influenza (HPAI) A(H5N8) virus has been reported with features of widely spread, an expanding host range, and cross-species transmission, attracting wide attention. The domestic duck plays a major role in the epidemiological cycle of the HPAI H5N8 virus, but little is known concerning innate immune responses during influenza infection in duck species. In this study, we used two wild-bird-origin viruses, H5N8 and H4N6, to conduct duck infection experiments, and detect the load of the two viruses, and retinoic acid-inducible gene I (RIG-I) and interferon β (IFN-β) in the host’s natural immune response. Through comparison, it is found that the expression levels of RIG-I and IFN-β are both fluctuating. The innate immunity starts rapidly within 6 h after infection and is inhibited by the virus to varying degrees. The expression of RIG-I and IFN-β decreased on 1–2 days post-infection (dpi). The HPAI H5N8 virus has a stronger inhibitory effect on RIG-I than the low pathogenic avian influenza (LPAI) H4N6 virus and is the strongest in the lungs. After infection with HPAI H5N8 virus, 2 dpi, viral RNA replicates in large amounts in the lungs. It has been proven that RIG-I and IFN-β play an important role in the innate immune response of ducks to HPAI H5N8 virus infection, especially in the lungs. The main battlefield of RIG-I and IFN-β after infection with the LPAI H4N6 virus is in the rectum. Both viruses have been effectively controlled after 7 dpi. These results will help to understand the transmission mechanisms of avian influenza virus in wild ducks and help effectively prevent and control avian influenza.

STUB1 activates antiviral response in zebrafish by promoting the expression of RIG-I

Spring viraemia of carp virus (SVCV) is a fierce pathogen causing high mortality in the common carp. At present, the treatment of spring viraemia of carp (SVC) is limited. Innate immunity is the host's first line of defense against microbial pathogens. Retinoic acid-inducible gene I (RIG-I) activation plays an essential role in the antiviral immune response. Virus infection can activate the RIG-I signaling and induce the production of interferon (IFN) and the expression of IFN-stimulated genes (ISGs). STUB1 (STIP1 homology and U-box containing protein 1) is a highly conserved cytoplasmic protein. This protein is known to exist widely in many biological systems and plays an important role in the process of immune regulation, but little is known in fish. To explore the immune function of STUB1 in fish, STUB1 gene was cloned from zebrafish and analyzed in this study. Zebrafish STUB1 showed 77% and 79% amino acid sequence homology with those from human and mouse, respectively. The amino acid sequence of zebrafish STUB1 contains three TPR domains and one U-box domain. Subcellular localization study revealed that STUB1 is located in the cytoplasm. And overexpression of zebrafish STUB1 resulted in the activation of the transcription of IFN1 and ISGs. Functional analysis showed that STUB1 was able to activate RIG-I signaling, and promote the expression of RIG-I, but STUB1 can degrade RIG-I in mammals. The proliferation of SVCV was significantly inhibited after the overexpression of STUB1 and N-terminal TPR domain of STUB1 in EPC cells. And through secondary structure analysis, overexpression of the mutant of STUB1 110 amino acid resulted in weakened antiviral ability. The expression of STUB1 was attenuated by poly(I:C) treatment and SVCV infection. In summary, this study demonstrated for the first time that STUB1 can induce the production of IFN, enhance the expression of ISGs by promoting the expression of RIG-I and inhibiting viral replication in fish. These findings may form the essential basis for the development of antiviral targets and drugs.

RIG-I Promotes Cell Viability, Colony Formation, and Glucose Metabolism and Inhibits Cell Apoptosis in Colorectal Cancer by NF-κB Signaling Pathway.

Background Retinoic acid-inducible gene-I (RIG-I) has crucial effects on various cancers, while RIG-I's detailed roles and mechanism in colorectal cancer (CRC) are uncovered. Methods qRT-PCR was used to detect the expression of RIG-I in CRC, adjacent nontumor specimens, and five cell lines. CCK-8, colony formation, and flow cytometry assays were conducted to study CRC cell viabilities. Extracellular acidification rates, lactate analysis, and ATP analysis were conducted to study the cell viabilities and glucose metabolism of CRC cells. Western blot is used to determine the proteins of NF-κBp65 in the nucleus and cytoplasm. Results This study revealed the upregulation of RIG-I in CRC tissues and cells and that high RIG-I expression was correlated with poor prognosis of CRC patients. In addition, silencing RIG-I inhibited cell viability as well as colony formation and promoted cell apoptosis in CRC cells, while RIG-I knockdown suppressed transplanted tumor growth and facilitated apoptosis in nude mice. Moreover, silencing RIG-I inhibited glucose metabolism by decreasing extracellular acidification rate, lactate production, adenosine triphosphate, and content of hypoxia-inducible factor 1α and pyruvate kinase isoform. 2.2-Deoxy-d-glucose, a glycolysis inhibitor, reduced the growth of CRC cells and promoted apoptosis in vitro and in vivo. In addition, RIG-I knockdown decreased NF-κB nuclear translocation. Besides, inhibiting NF-κB effectively eliminated RIG-I overexpression roles in cell viability and glucose metabolism in CRC cells. Conclusion In summary, this study revealed that RIG-I mediated CRC cell proliferation, apoptosis, and glucose metabolism at least partly by NF-κB signaling pathway.

The Priming Potential of Interferon Lambda-1 for Antiviral Defense in the Oral Mucosa.

The oral mucosa is one of the first lines of the innate host defense system against microbial invasion. Interferon (IFN) lambda-1 (IFN-λ1), a type III IFN, exhibits type I IFN-like antiviral activity. In contrast to ubiquitously expressed type I IFN receptors, IFN-λ receptor 1 (IFN-λR1), which has higher affinity for type III IFNs than low-affinity interleukin (IL)-10 receptor 2, is mainly expressed on epithelial cells. Although IFN-λ1 has been shown to exert antiviral effects in the respiratory tract, gastrointestinal tract, and skin, the regulation of type III IFN receptor expression and its functions in the oral mucosa remain unclear. We herein showed the expression of IFN-λR1 in human gingival keratinocytes. The expression of IL-6, angiotensin-converting enzyme 2 (a critical molecule for severe acute respiratory syndrome coronavirus 2 infection), and IL-8 in human primary gingival keratinocytes (HGK) were significantly higher following treatments with either type I IFN (IFN-β) or type II IFN (IFN-γ) than with IFN-λ1. However, the IFN-λ1 treatment strongly induced toll-like receptor (TLR) 3 and retinoic acid-inducible gene I (RIG-I), which mainly recognize viral nucleic acids, via the STAT1-mediated pathway. Furthermore, a stimulation with a RIG-I or TLR3 agonist promoted the production of IL-6, IL-8, and IFN-λ in HGK, which was significantly enhanced by a pretreatment with IFN-λ1. These results suggest that IFN-λ1 may contribute to the activation of innate immune responses to oral viral infections by up-regulating the expression of RIG-I and TLR3 and priming their functions in keratinocytes.Supplementary InformationThe online version contains supplementary material available at 10.1007/s10753-022-01624-1.

Klotho deficiency intensifies hypoxia-induced expression of IFN-α/β through upregulation of RIG-I in kidneys.

Hypoxia is a common pathway to the progression of end-stage kidney disease. Retinoic acid-inducible gene I (RIG-I) encodes an RNA helicase that recognizes viruses including SARS-CoV2, which is responsible for the production of interferon (IFN)-α/β to prevent the spread of viral infection. Recently, RIG-I activation was found under hypoxic conditions, and klotho deficiency was shown to intensify the activation of RIG-I in mouse brains. However, the roles of these functions in renal inflammation remain elusive. Here, for in vitro study, the expression of RIG-I and IFN-α/β was examined in normal rat kidney (NRK)-52E cells incubated under hypoxic conditions (1% O2). Next, siRNA targeting RIG-I or scramble siRNA was transfected into NRK52E cells to examine the expression of RIG-I and IFN-α/β under hypoxic conditions. We also investigated the expression levels of RIG-I and IFN-α/β in 33 human kidney biopsy samples diagnosed with IgA nephropathy. For in vivo study, we induced renal hypoxia by clamping the renal artery for 10 min in wild-type mice (WT mice) and Klotho-knockout mice (Kl-/- mice). Incubation under hypoxic conditions increased the expression of RIG-I and IFN-α/β in NRK52E cells. Their upregulation was inhibited in NRK52E cells transfected with siRNA targeting RIG-I. In patients with IgA nephropathy, immunohistochemical staining of renal biopsy samples revealed that the expression of RIG-I was correlated with that of IFN-α/β (r = 0.57, P<0.001, and r = 0.81, P<0.001, respectively). The expression levels of RIG-I and IFN-α/β were upregulated in kidneys of hypoxic WT mice and further upregulation was observed in hypoxic Kl-/- mice. These findings suggest that hypoxia induces the expression of IFN-α/β through the upregulation of RIG-I, and that klotho deficiency intensifies this hypoxia-induced expression in kidneys.

5-Hydroxymethylfurfural Enhances the Antiviral Immune Response in Macrophages through the Modulation of RIG-I-Mediated Interferon Production and the JAK/STAT Signaling Pathway.

5-Hydroxymethylfurfural (5-HMF) exists in a wide range of sugar-rich foods and traditional Chinese medicines. The role of 5-HMF in antiviral innate immunity and its mechanism have not been reported previously. In this study, we reveal for the first time that 5-HMF upregulates the production of retinoic acid-inducible gene I (RIG-I)-mediated type I interferon (IFN) as a response to viral infection. IFN-β and IFN-stimulated chemokine gene expressions induced by the vesicular stomatitis virus (VSV) are upregulated in RAW264.7 cells and primary peritoneal macrophages after treatment with 5-HMF, a natural product that appears to inhibit the efficiency of viral replication. Meanwhile, 5-HMF-pretreated mice show enhanced innate antiviral immunity, increased serum levels of IFN-β, and reduced morbidity and viral loads upon infection with VSV. Thus, 5-HMF can be seen to have a positive effect on enhancing type I IFN production. Mechanistically, 5-HMF upregulates the expression of RIG-I in macrophages, resulting in an acceleration of the RIG-I signaling pathway activation. Additionally, STAT1 and STAT2 phosphorylations, along with the expression of IFN-stimulated chemokine genes induced by IFN-α/β, were also enhanced in macrophages cotreated with 5-HMF. In summary, these findings indicate that 5-HMF not only can induce type I IFN production but also can enhance IFN-JAK/STAT signaling, leading to a novel immunomodulatory mechanism against viral infection. In conclusion, our study reveals a previously unrecognized effect of 5-HMF in the antiviral innate immune response and suggests new potential of utilizing 5-HMF for controlling viral infection.

Modulation of SF3B1 in the pre-mRNA spliceosome induces a RIG-I-dependent type I IFN response

Nucleic acid–sensing pathways play critical roles in innate immune activation through the production of type I interferon (IFN-I) and proinflammatory cytokines. These factors are required for effective antitumor immune responses. Pharmacological modulators of the pre-mRNA spliceosome splicing factor 3b subunit 1 (SF3B1) are under clinical investigation as cancer cytotoxic agents. However, potential roles of these agents in aberrant RNA generation and subsequent RNA-sensing pathway activation have not been studied. In this study, we observed that SF3B1 pharmacological modulation using pladienolide B (Plad B) induces production of aberrant RNA species and robust IFN-I responses via engagement of the dsRNA sensor retinoic acid–inducible gene I (RIG-I) and downstream interferon regulatory factor 3. We found that Plad B synergized with canonical RIG-I agonism to induce the IFN-I response. In addition, Plad B induced NF-κB responses and secretion of proinflammatory cytokines and chemokines. Finally, we showed that cancer cells bearing the hotspot SF3B1K700E mutation, which leads to global aberrant splicing, had enhanced IFN-I response to canonical RIG-I agonism. Together, these results demonstrate that pharmacological modulation of SF3B1 in cancer cells can induce an enhanced IFN-I response dependent on RIG-I expression. The study suggests that spliceosome modulation may not only induce direct cancer cell cytotoxicity but also initiate an innate immune response via activation of RNA-sensing pathways.

Human Eosinophils Reduce Viral Titer, Secrete IL-8, and Increase RIG-I Expression in Response to Influenza A H1N1 pdm09.

Eosinophils participate in the immune response against many pathogens, including viruses. Since mouse eosinophils are susceptible to influenza A virus infection and possess antiviral activity, we evaluated the expression of sialic acid residues in human eosinophils and their response against influenza virus in vitro. We demonstrated that human eosinophils express α2,6- and α2,3-linked sialic acid, and drastically reduced influenza virus titer. After influenza virus exposure, eosinophils upregulated retinoic acid-inducible gene I (RIG-I) mRNA expression, but no other pattern recognition receptors. Finally, high concentrations of interleukin-8 (IL-8) were found in influenza virus-exposed eosinophil cultures. These data suggest that human eosinophils possess antiviral activity and may play a role in the innate immune response to influenza virus.

Knock-in of the duck retinoic acid-inducible gene I (RIG-I) into the Mx gene in DF-1 cells enables both stable and immune response-dependent RIG-I expression

Waterfowls, such as ducks, are natural hosts of avian influenza virus (AIV) and can genetically limit the pathogenicity. On the other hand, some AIV strains cause severe pathogenicity in chickens. It is suggested that differences in the pathogenicity of AIV infection between waterfowls and chickens are related to the expression of retinoic acid-inducible gene I (RIG-I), a pattern recognition receptor that chickens evolutionally lack. Here, we knocked-in the duck RIG-I bearing the T2A peptide sequence at the 3′ region of the Mx, an interferon-stimulated gene (ISG), in chicken embryo fibroblast cells (DF-1) using the precise integration into target chromosome (PITCh) system to control the duck RIG-I expression in chickens. The expression patterns of the duck RIG-I were then analyzed using qPCR. The knocked-in DF-1 cells expressed RIG-I via the stimulation of IFN-β and poly(I:C) in a dose-dependent manner. Moreover, poly(I:C) stimulation in the knocked-in DF-1 cells upregulated RIG-I-like receptor (RLR) family signaling pathway-related genes IFN-β, OASL, and IRF7. The IFN-β-dependent expression of RIG-I and upregulation of IFN-β in the poly(I:C) stimulation demonstrated a positive-feedback loop via RIG-I, usually evident in ducks. Overall, this novel strategy established RIG-I-dependent immune response in chickens without overexpression of RIG-I and disruption of the host genes.

Preferential In Vitro and In Vivo Induction of Endogenous Retrovirus Transcription from a Monoallelic Chromosome 7q in Decitabine (DAC) Treated AML Blasts

Background: Hypomethylating agents (HMAs) show an encouraging but not yet well-understood activity in AML/MDS patients (pts) with adverse cytogenetics, such as -7/7q-, often embedded in a complex, monosomal karyotype (Lübbert et al., Haematologica 2012). Integrative methylome/transcriptome studies have provided evidence for aberrant hypermethylation/silencing on monoallelic gene loci, including tumor suppressor genes (TSGs, Kotini et al., Nat. Biotechnol. 2015). Recently, an alternative mechanism of HMA action was described (Roulois et al., Chiappinelli et. al, Cell 2015): induction of silenced endogenous retroviral (ERV) dsRNA transcription, resulting in activation of an interferon-mediated, antitumor immune response (“viral mimicry”). To unravel in vivo HMA activity in different (cyto)genetic backgrounds, we hypothesized that in AML haploinsufficient for 7q, transcriptional repression of monoallelic genes in this region may be preferentially reversed by HMA treatment.Materials and Methods: For identification of genes induced by HMAs, two AML cell lines were selected, ELF-153 (ELF) and UCSD-AML1 (AML1): copy number variations were analyzed by Genome-Wide Human SNP Array 6.0 (Affymetrix), and revealed no monosomies but loss of 7p; in AML1 loss of one entire chromosome 7 was the only numerical aberration. Cells were treated with DAC at equitoxic and -effective concentrations for 4 days, harvested on day 5. Total RNA was depleted of ribosomal RNA (Ribo-Zero rRNA Removal Kit, Illumina) and cDNA libraries (strand-specific, 50 bp) were sequenced with ≥33 million paired-end reads, read quality checked with FastQC (≥95% >Q30). Alignment to the reference genome hg19 was performed with TopHat2, read-counting with HTseq, differential expression testing with DESeq2 (protein-coding transcripts only). Selected target genes were validated by RT-qPCR. Peripheral blood mononuclear cells (PBMC) were collected from 9 AML pts (5 pts with 7q-, 4 cytogenetically normal [CN] pts) treated with DAC (i.v. 20 mg/m2 for 5 days) within the DECIDER trial (NCT00867672). Leukemic blasts before treatment and at day 8 were isolated using automatic magnetic sorting of cells labeled with anti-human CD34 and CD117 MACS microbeads.Results: DAC treatment of AML1 and ELF cells resulted in massive transcriptome changes in both cell lines, with 1,704 genes induced in AML1 (total of 3,583) and 1,206 in ELF (total of 1,867; adjusted FDR <0.01). Comparing DAC-induced expression changes only of genes on 7q, we identified 43 genes significantly differentially expressed only in AML1 cells (up: 15), 24 only in ELF (up: 21), and 19 in both (up: 12). The 15 genes on 7q that were selectively upregulated in AML1 included 3 with TSG features: zyxin (ZYX; up 6.0-fold), homeodomain interacting protein kinase 2 (HIPK2; up 3.8-fold), and high mobility group box transcription factor 1 (HBP1, up 3.1-fold). However, the most heavily upregulated transcripts in AML1 were the endogenous retrovirus 3 group member 1 (ERV3-1 ; up 86.4-fold in AML1 vs 14.3-fold in ELF) and the neighboring zinc finger 117 (ZNF117; up 119.4-fold in AML1vs. 6.5-fold in ELF), both transcribed from the ERV3-1 promoter. ERV3-1 induction was accompanied by induction of the interferon type I response genes retinoic acid inducible gene I (RIG-I) and interferon regulatory factor 7 (IRF7) in both cell lines. Therefore, we next interrogated induction of these genes by DAC in vivo, utilizing serially sorted, matched primary AML blasts (purified before and 3 days after DAC administration). Stronger induction of ERV3-1 and RIG-I mRNA was seen in the 7q- pts compared to the CN pts: By qRT-PCR, median ERV3-1 and RIG-I expression was induced 1.5- and 1.4-fold, respectively, in the pts with 7q- (range: 1.1-2.1 and 1.0-3.2), 1.1- and 0.7-fold, respectively, in the CN pts (range: 0.8-1.7 and 0.3-0.9).Conclusions: We successfully developed an unbiased RNA-seq approach of AML cell lines either mono- or bi-allelic for 7q, demonstrating that DAC treatment preferential upregulates several monoallelic TSGs, and massively activates the ERV3-1 gene (implicated in “viral mimicry”). Induction of ERV3-1 and the dsRNA sensor RIG-I was validated in vivo, and preferentially seen in 7q- AML purified primary blasts. Thus, under clinically established treatment conditions, both the ERV and an interferon response gene can be activated by DAC, supporting a combination with immunotherapy. DisclosuresLübbert:Janssen-Cilag: Other: Travel Funding, Research Funding; Ratiopharm: Other: Study drug valproic acid; Celgene: Other: Travel Funding.

Reciprocal regulation of RIG-I and XRCC4 connects DNA repair with RIG-I immune signaling

The RNA-sensing pathway contributes to type I interferon (IFN) production induced by DNA damaging agents. However, the potential involvement of RNA sensors in DNA repair is unknown. Here, we found that retinoic acid-inducible gene I (RIG-I), a key cytosolic RNA sensor that recognizes RNA virus and initiates the MAVS-IRF3-type I IFN signaling cascade, is recruited to double-stranded breaks (DSBs) and suppresses non-homologous end joining (NHEJ). Mechanistically, RIG-I interacts with XRCC4, and the RIG-I/XRCC4 interaction impedes the formation of XRCC4/LIG4/XLF complex at DSBs. High expression of RIG-I compromises DNA repair and sensitizes cancer cells to irradiation treatment. In contrast, depletion of RIG-I renders cells resistant to irradiation in vitro and in vivo. In addition, this mechanism suggests a protective role of RIG-I in hindering retrovirus integration into the host genome by suppressing the NHEJ pathway. Reciprocally, XRCC4, while suppressed for its DNA repair function, has a critical role in RIG-I immune signaling through RIG-I interaction. XRCC4 promotes RIG-I signaling by enhancing oligomerization and ubiquitination of RIG-I, thereby suppressing RNA virus replication in host cells. In vivo, silencing XRCC4 in mouse lung promotes influenza virus replication in mice and these mice display faster body weight loss, poorer survival, and a greater degree of lung injury caused by influenza virus infection. This reciprocal regulation of RIG-I and XRCC4 reveals a new function of RIG-I in suppressing DNA repair and virus integration into the host genome, and meanwhile endues XRCC4 with a crucial role in potentiating innate immune response, thereby helping host to prevail in the battle against virus.